Streptococcus pneumoniae capsule polysaccharide and its immunogenic conjugate

Abstract

FIELD: biotechnology.SUBSTANCE: immunogenic composition for preventing an infection, disease or condition caused by serotype 20 Streptococcus pneumoniae is described, containing an effective amount of a conjugate of a capsular polysaccharide from serotype 20 Streptococcus pneumoniae – CRM197 protein and a pharmaceutically acceptable excipient, carrier or diluent, wherein said activated capsular polysaccharide intended for conjugation with the protein has an oxidation state of 4–16 and a molecular weight of 400–800 kDa. Also the use of said immunogenic composition for preventing an infection, disease or condition caused by serotype 20 Streptococcus pneumoniae is described.EFFECT: protection against Streptococcus pneumoniae serotype 20.6 cl, 15 tbl, 2 ex

Description

[0001] [TECHNICAL AREA]

[0002] This application claims the right of priority based on Korean Patent Application No. 10-2018-0045245 filed on April 18, 2018, Korean Patent Application No. 10-2018-0045246 filed on April 18, 2018, Korean Patent Application No. 10-2018-0045247 filed on April 18, 2018, and Korean Patent Application No. 10-2018-0045248 filed on April 18, 2018, and the entire contents of these applications disclosed in the description and drawings are incorporated herein by reference.

[0003] The present invention relates to an immunogenic composition and a vaccine for Streptococcus pneumoniae, and more particularly, the present invention relates to an immunogenic composition and a vaccine that comprises a Streptococcus pneumoniae capsular polysaccharide-carrier protein conjugate.

[0004] [STATE OF THE ART]

[0005] Streptococcus pneumoniae is the main bacteria that causes pneumonia. In addition, it causes invasive diseases such as septicemia, bacteremia, meningitis, etc. According to the National Statistics Office [cause of death statistics for 2014], the mortality rate from pneumonia in 2014 was 23.7 people per hundred thousand, compared to a 2.8-fold increase in 2015, and the mortality rate from pneumonia continues to rise continuously. In addition, according to a WHO report published in 2012, in 2008, 476,000 HIV-negative children under 5 years of age worldwide died of Streptococcus pneumoniae infection, accounting for 5% of deaths in children under 5 years of age. Pneumococci are divided into more than 90 serotypes based on the structural and immunological characteristics of the capsular polysaccharide, which is the main virulence factor, located around their outer surface (in the cell membrane).

[0006] To prevent diseases caused by Streptococcus pneumoniae, Dr. Robert Austrian developed a 14-valent polysaccharide vaccine in 1977. Further development led to the development of a 23-valent polysaccharide vaccine. The polyvalent pneumococcal polysaccharide vaccine has been shown to be effective in preventing diseases caused by Streptococcus pneumoniae in the elderly and high-risk patients. However, infants and children lack an immune response to most Streptococcus pneumoniae polysaccharides, as their immune response is not T-cell dependent. Therefore, a conjugate vaccine based on Streptococcus pneumoniae capsular polysaccharide and a carrier protein was developed that can induce a T-cell-dependent response.

[0007] 7-valent conjugate vaccine Streptococcus pneumoniae (Prevenar ®) contains capsular polysaccharides isolated from the 7 most common serotypes 4, 6B, 9V, 14, 18C, 19F and 23F. It has been shown to be highly immunogenic and effective against invasive pneumococcal disease and otitis media in infants and children, thus it was initially approved in the United States in 2000. Then, Prevenar 13®, a 13-valent conjugate vaccine to which 6 serotypes 1, 3, 5, 6A, 7F, 19A were added, and Synflorix, a 10-valent conjugate vaccine to which 3 serotypes 1, 5, 7F were added, were successively developed and the incidence of invasive disease caused by Streptococcus pneumoniae was further reduced. However, the serotype changes brought about by the introduction of Prevenar, Prevenar 13 and Synflorix and the overall decline in the incidence of disease caused by vaccine-containing serotypes particularly highlight the importance of non-vaccine serotypes, which were previously of relatively low importance.

[0008] In particular, in North America and Brazil, the incidence of invasive pneumococcal diseases caused by Streptococcus pneumoniae serotype 20 has increased (see, for example, [Kendall B. et al., Vaccine. 34:474–478, 2016], [Yildirim I. et al., Pediatr Infect Dis J. 31(10): 1016–1021, 2012], or [Caierão J. et al., PLoS ONE 9(10): e111129, 2014]). Furthermore, in the CASPER study in Canada, an increase in serotypes 8 and 12F, as well as serotype 19A, was observed (Sá-Leão A. et al., J Clin Microbiol., 49(4): 1369-75, 2011). A recent study reported that in Norway and Israel, after the introduction of Prevenar 13, there was an increase in cases of disease caused by non-vaccine serotypes, and examples of non-vaccine serotypes included 23A, 23B, 12F, 15A / 15B / 15C, 31, 33F, 7C, and 8 (Martin J., Pediatr Infect Dis J., 33(11):e286-90, 2014).

[0009] Despite the steady increase in the incidence of pneumococcal diseases caused by Streptococcus pneumoniae serotypes 2, 9N, 17F and / or 20, studies on effective measures to prevent or treat infections caused by these serotypes have not been conducted.

[0010] There is thus an increasing need for immunogenic conjugates and immunogenic compositions directed to non-vaccine serotypes containing serotypes that are included in polyvalent polysaccharide vaccines but not included in conjugate vaccines to provide broader spectrum protection.

[0011] [DESCRIPTION]

[0012] [TECHNICAL PROBLEM]

[0013] Accordingly, the problem to be solved by the present invention is to provide a polyvalent vaccine that can provide a broad spectrum of protection.

[0014] In addition, an immunogenic composition containing a new serotype that has not previously been included in a conjugate vaccine is also provided.

[0015] Furthermore, the problem to be solved by the present invention is to provide a pneumococcal conjugate vaccine that provides an excellent antibody titer.

[0016] [TECHNICAL SOLUTION]

[0017] In one embodiment of the present invention, an immunogenic composition is provided, comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, comprising a capsular polysaccharide isolated from one or more Streptococcus pneumoniae serotypes selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F; and a carrier protein conjugated to each capsular polysaccharide.

[0018] In one embodiment, an immunogenic composition is provided comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein if the immunogenic composition comprises a polysaccharide isolated from serotype 2, the polysaccharide having a molecular weight of 100 to 400 kDa is activated and binds the carrier protein to form a conjugate, or

[0019] if the immunogenic composition contains a polysaccharide isolated from serotype 17F, then the polysaccharide with a molecular weight of 400 to 900 kDa is activated and binds the carrier protein to form a conjugate, or

[0020] If the immunogenic composition contains a polysaccharide isolated from serotype 20, the polysaccharide with a molecular weight of 400 to 800 kDa is activated and binds the carrier protein to form a conjugate.

[0021] In one embodiment of the present invention, there is provided an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein if the immunogenic composition comprises a polysaccharide isolated from serotype 2, then the immunogenic conjugate comprising a polysaccharide isolated from serotype 2 has a molecular weight of 1,000 to 16,000 kDa, or

[0022] if the immunogenic composition contains a polysaccharide isolated from serotype 17F, then the immunogenic conjugate containing the polysaccharide isolated from serotype 17F has a molecular weight of 300 to 4500 kDa, or

[0023] if the immunogenic composition contains a polysaccharide isolated from serotype 20, then the immunogenic conjugate containing the polysaccharide isolated from serotype 20 has a molecular weight of 1000 to 4000 kDa.

[0024] In one embodiment of the present invention, there is provided an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, characterized in that the carrier protein is TT (tetanus toxoid) or CRM 197 , and preferably the immunogenic composition containing only one serotype may contain CRM 197 as a carrier protein.

[0025] In one embodiment of the present invention, there is provided an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein if the immunogenic composition comprises a polysaccharide isolated from serotype 2, then the ratio of serotype 2 capsular polysaccharide to carrier protein in the immunogenic conjugate (polysaccharide / protein, (w / w)) is from 0.5 to 2.0, or

[0026] if the immunogenic composition contains a polysaccharide isolated from serotype 17F, then the ratio of the capsular polysaccharide of serotype 17F to the carrier protein in the immunogenic conjugate (polysaccharide / protein, (w / w)) is from 0.5 to 18, or

[0027] if the immunogenic composition contains a polysaccharide isolated from serotype 20, then the ratio of the capsular polysaccharide of serotype 20 to the carrier protein in the immunogenic conjugate (polysaccharide / protein, (w / w)) is from 1 to 5.

[0028] In one embodiment of the present invention, there is provided an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein in the molecular weight distribution from 20 to 60% of the conjugate is within 0.3 Kd when using a CL-4B column in the case of an immunogenic conjugate comprising a polysaccharide isolated from serotype 2, or

[0029] in a molecular weight distribution of 15 to 60% of the conjugate is within 0.3 Kd when using a CL-4B column in the case of an immunogenic conjugate containing a polysaccharide isolated from serotype 17F, or

[0030] In the molecular weight distribution of 70 to 90% of the conjugate, it is within 0.3 Kd when using the CL-4B column in the case of the immunogenic conjugate containing the polysaccharide isolated from serotype 20.

[0031] In one embodiment of the present invention, there is provided an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein

[0032] in the conjugate, the oxidation state of the conjugated polysaccharide is from 2 to 18 in the case of an immunogenic conjugate containing a polysaccharide isolated from serotype 2, or

[0033] in the conjugate, the oxidation state of the conjugated polysaccharide is from 1 to 22 in the case of an immunogenic conjugate containing a polysaccharide isolated from serotype 17F, or

[0034] In the conjugate, the oxidation state of the conjugated polysaccharide is from 4 to 16 in the case of an immunogenic conjugate containing a polysaccharide isolated from serotype 20.

[0035] In one embodiment of the present invention, there is provided an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein the immunogenic composition is a composition in which polysaccharides isolated from 15 serotypes distinct from each other are conjugated to corresponding carrier proteins, and

[0036] serotypes include 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F and 23F, and

[0037] serotypes are conjugated with CRM 197.

[0038] In one embodiment of the present invention, there is provided an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein the immunogenic composition is a composition in which polysaccharides isolated from 23 serotypes distinct from each other are conjugated to corresponding carrier proteins, and

[0039] serotypes include 1, 2, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, and

[0040] Among these serotypes, capsular polysaccharides isolated from serotypes 3 and 5 are conjugated to the TT carrier protein, and capsular polysaccharides isolated from serotypes 1, 2, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F are conjugated to the CRM197 carrier protein.

[0041] In one embodiment of the present invention, there is provided an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein the immunogenic composition is a composition in which polysaccharides isolated from 24 serotypes distinct from each other are conjugated to corresponding carrier proteins, and

[0042] serotypes include 2, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, and

[0043] capsular polysaccharides isolated from serotypes 1 and 5 are conjugated to the TT carrier protein, and capsular polysaccharides isolated from serotypes 2, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F are conjugated to the CRM197 carrier protein.

[0044] In one embodiment of the present invention, an immunogenic composition is provided comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, which comprises a physiologically acceptable filler.

[0045] In one embodiment of the present invention, an immunogenic composition is provided comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein the immunogenic composition is a vaccine.

[0046] In one embodiment of the present invention, a method for producing an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate comprising

[0047] (a) a step of fermenting and dissolving a bacterial cell producing a capsular polysaccharide isolated from one or more serotypes selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F of Streptococcus pneumoniae;

[0048] (b) a step of purifying the capsular polysaccharide isolated from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F of Streptococcus pneumoniae in a dissolved cell;

[0049] (c) a step of bringing the purified polysaccharide into contact with an oxidizing agent to activate it; and

[0050] (d) a step of combining the activated polysaccharide with a carrier protein to obtain a Streptococcus pneumoniae polysaccharide-protein conjugate bound to a carrier protein.

[0051] In one embodiment of the present invention, the production method may further comprise a step of hydrolyzing the purified capsular polysaccharide of Streptococcus pneumoniae to give it the desired size before step (c) in the case of capsular polysaccharides isolated from serotypes 2 and 17F.

[0052] In one embodiment of the present invention, a method for producing an immunogenic composition containing a Streptococcus pneumoniae polysaccharide-protein conjugate is provided, characterized in that the carrier protein used for combining in step (d) forms a conjugate with an activated polysaccharide by reacting with one or more reducing agents selected from the group consisting of cyanoborohydride, borane-pyridine and a borohydride ion exchange resin.

[0053] In one embodiment of the present invention, a method for producing an immunogenic composition containing a Streptococcus pneumoniae polysaccharide-protein conjugate is provided, characterized in that step (c) involves reacting using 0.01 ~ 0.22 μg of periodate per 1 μg of polysaccharide at a temperature of 20 to 25°C for a period of 15 to 20 hours.

[0054] In one embodiment of the present invention, a Streptococcus pneumoniae polysaccharide-protein conjugate for preventing or treating Streptococcus pneumoniae infection is provided, obtained by the said method.

[0055] In one embodiment of the present invention, there is provided a method for preventing or treating an infection caused by serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and / or 33F of Streptococcus pneumoniae in a subject,

[0056] by administering an effective dose of an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate containing

[0057] a capsular polysaccharide isolated from one or more serotypes of Streptococcus pneumoniae selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F; and

[0058] one or 2 or more carrier proteins conjugated to corresponding capsular polysaccharides, to a subject. "And / or" means "and" or "or".

[0059] The method can prevent or treat infections caused by one or more serotypes selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, or infection with one or more serotypes can occur, and the method can prevent infection with 15 serotypes, or the method can prevent infection with 23 serotypes, or the method can prevent infection with 24 serotypes.

[0060] In one example of the present invention, for the prevention or treatment of pneumococcal infection, the use of a Streptococcus pneumoniae polysaccharide-protein conjugate containing

[0061] a capsular polysaccharide isolated from one or more serotypes of Streptococcus pneumoniae selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F; and

[0062] one or 2 or more carrier proteins conjugated to corresponding capsular polysaccharides.

[0063] In one example of the present invention, there is provided an immunogenic conjugate of serotype 2 Streptococcus pneumoniae comprising a capsular polysaccharide of serotype 2 isolated from Streptococcus pneumoniae; and a carrier protein associated with the capsular polysaccharide.

[0064] Serotype 2 polysaccharide with a molecular weight of 100 to 400 kDa can be activated and bind a carrier protein to form a conjugate.

[0065] In one example of the present invention, the immunogenic conjugate may have a molecular weight of 1,000 to 16,000 kDa, and, for example, the carrier protein may be a CRM 197 .

[0066] In one example of the present invention, the ratio of serotype 2 capsular polysaccharide to carrier protein in the immunogenic conjugate (w / w) may be from 0.5 to 2.0.

[0067] 20 to 60% of the serotype 2 immunogenic conjugate can be within 0.3 Kd using the CL-4B column. Another example provides an immunogenic conjugate of Streptococcus pneumoniae serotype 2 having an oxidation state of 2 to 18.

[0068] If the polysaccharide of Streptococcus pneumoniae serotype 2 is oxidized by adding 0.02 to 0.12 μg of periodate per 1 μg of sugar and conjugated with a protein, the molecular weight of the conjugate may be 1000 ~ 16000 kDa, and 20 ~ 60% of the conjugate may be within the specified range (0.3 kd or less) of the molecular weight distribution, and the polysaccharide / protein ratio may be 0.5 to 2.0.

[0069] As one example of the present invention, an immunogenic composition comprising an immunogenic conjugate and a physiologically acceptable excipient can be provided.

[0070] As another example of the present invention, a vaccine containing an immunogenic composition can be provided.

[0071] As another example of the present invention, there can be provided a method for producing an immunogenic conjugate of serotype 2 Streptococcus pneumoniae, comprising

[0072] (a) a step of fermenting and dissolving a bacterial cell producing capsular polysaccharide of serotype 2 Streptococcus pneumoniae;

[0073] (b) a step of purifying the capsular polysaccharide of serotype 2 Streptococcus pneumoniae in the dissolved cell;

[0074] (c) a step of hydrolyzing the purified capsular polysaccharide of serotype 2 Streptococcus pneumoniae to give the polysaccharide a desired size;

[0075] (d) a step of reacting the polysaccharide of the desired size obtained in step (c) to activate the polysaccharide; and

[0076] (e) a step of combining the activated polysaccharide with a carrier protein to obtain a conjugate of Streptococcus pneumoniae serotype 2 capsular polysaccharide bound to a carrier protein.

[0077] The carrier protein used for the combination in step (e) can form a conjugate with the activated polysaccharide by reacting with a reducing agent.

[0078] Step (d) may include a reaction process using 0.02~0.12 μg of periodate per 1 μg of polysaccharide at a temperature of 20 to 25°C for a period of 15 to 20 hours.

[0079] The activated polysaccharide, which is combined with the carrier protein in step (e), may have a molecular weight of 100 to 400 kDa.

[0080] A method for producing an immunogenic conjugate of Streptococcus pneumoniae serotype 2, wherein the carrier protein is CRM197, may be provided.

[0081] The immunogenic conjugate may have a molecular weight of 1000 to 16000 kDa.

[0082] In one example, the initial ratio of the administered activated serotype 2 capsular polysaccharide to the carrier protein (carrier protein:polysaccharide) may be from 0.5 to 2:1.

[0083] As another example, a method for producing an immunogenic conjugate of Streptococcus pneumoniae serotype 2 can be provided, wherein at least 20 to 60% of the immunogenic conjugate is within 0.3 Kd when using a CL-4B column.

[0084] As one example of the present invention, an immunogenic conjugate obtained by the said method can be provided.

[0085] As one example of the present invention, an immunogenic composition may be provided comprising an immunogenic conjugate obtained by the said method and a physiologically acceptable filler.

[0086] As another example, a vaccine containing an immunogenic composition may be provided.

[0087] The present invention provides an immunogenic conjugate of serotype 9N Streptococcus pneumoniae, comprising a capsular polysaccharide of serotype 9N isolated from Streptococcus pneumoniae; and a carrier protein associated with the capsular polysaccharide.

[0088] In one example of the present invention, a serotype 9N polysaccharide with a molecular weight of 200 to 700 kDa can be activated and bind a carrier protein to form a conjugate.

[0089] The immunogenic conjugate may have a molecular weight of 500 to 4000 kDa, and the carrier protein may be a CRM 197 .

[0090] The ratio of serotype 9N capsular polysaccharide to the carrier protein in the immunogenic conjugate (w / w) is 0.1 to 5, and may preferably be 0.5~2.5.

[0091] In one example, 15 to 60% of the immunogenic conjugate can be within 0.3 Kd when using a CL-4B column.

[0092] In one example, the conjugate may have an oxidation state of from 2 to 19.

[0093] In one example of the present invention, if the polysaccharide of Streptococcus pneumoniae serotype 9N is oxidized by adding 0.02~0.19 μg of periodate per 1 μg of sugar and conjugated with a protein, the molecular weight of the conjugate can be 500~4000 kDa, and 15~60% of the conjugate can be within a predetermined range (0.3 kd or less) of the molecular weight distribution, and the polysaccharide / protein ratio can be 0.5~2.5.

[0094] As one example of the present invention, an immunogenic composition comprising an immunogenic conjugate and a physiologically acceptable excipient can be provided.

[0095] As another example of the present invention, a vaccine containing an immunogenic composition can be provided.

[0096] As another example of the present invention, there may be provided a method for producing an immunogenic conjugate of serotype 9N Streptococcus pneumoniae, comprising

[0097] (a) a step of fermenting and dissolving a bacterial cell producing capsular polysaccharide of serotype 9N Streptococcus pneumoniae;

[0098] (b) a step of purifying the capsular polysaccharide of serotype 9N Streptococcus pneumoniae in the dissolved cell;

[0099] (c) a step of bringing the polysaccharide into contact with an oxidizing agent to activate it; and

[0100] (d) a step of combining the activated polysaccharide with a carrier protein to obtain a conjugate of Streptococcus pneumoniae serotype 9N capsular polysaccharide bound to a carrier protein.

[0101] A method for producing an immunogenic conjugate of serotype 9N Streptococcus pneumoniae may be provided, characterized in that the carrier protein used for combining in step (d) is brought into contact with a reducing agent to produce a conjugate with an activated polysaccharide.

[0102] Step (c) may include a reaction process using 0.02 ~ 0.19 μg of periodate per 1 μg of polysaccharide at a temperature of 20 to 25°C for a period of 15 to 20 hours.

[0103] The polysaccharide reacted with the oxidizing agent in step (c) may have a molecular weight of 400 to 900 kDa.

[0104] The activated polysaccharide combined with the carrier protein in step (d) may have a molecular weight of 200-700 kDa.

[0105] The immunogenic conjugate may have a molecular weight of 500 to 4000 kDa.

[0106] The initial ratio of the administered activated capsular polysaccharide of serotype 9N to the carrier protein (carrier protein:polysaccharide) can be from 0.5 to 2.5:1.

[0107] In one example, at least 15% to 60% of the immunogenic conjugate may be within 0.3 Kd when using a CL-4B column.

[0108] As one example of the present invention, an immunogenic conjugate obtained by the said method can be provided. As one example of the present invention, an immunogenic composition comprising the immunogenic conjugate obtained by the said method and a physiologically acceptable excipient can be provided. As another example, a vaccine comprising the immunogenic composition can be provided.

[0109] In one example of the present invention, there is provided an immunogenic conjugate of serotype 17F Streptococcus pneumoniae, comprising a capsular polysaccharide of serotype 17F isolated from Streptococcus pneumoniae; and a carrier protein associated with the capsular polysaccharide.

[0110] Serotype 17F polysaccharide with a molecular weight of 400 to 900 kDa can be activated and bind a carrier protein to form a conjugate.

[0111] In one example of the present invention, the immunogenic conjugate may have a molecular weight of 300 to 4500 kDa, and, for example, the carrier protein may be a CRM 197 .

[0112] In one example of the present invention, the ratio of serotype 17F capsular polysaccharide to carrier protein in the immunogenic conjugate (w / w) may be from 0.5 to 18.

[0113] From 15% to 60% of the immunogenic conjugate of serotype 17F can be detected within 0.3 Kd using the CL-4B column. Another example provides an immunogenic conjugate of serotype 17F Streptococcus pneumoniae having an oxidation state of 1 to 22.

[0114] If the polysaccharide of Streptococcus pneumoniae serotype 17F is oxidized by adding 0.01 to 0.22 μg of periodate per 1 μg of sugar and conjugated with a protein, the molecular weight of the conjugate may be 300 to 4500 kDa, and 15~60% of the conjugate may be within the specified range (0.3 kd or less) of the molecular weight distribution, and the polysaccharide / protein ratio may be 0.5 to 18.

[0115] As one example of the present invention, an immunogenic composition comprising an immunogenic conjugate and a physiologically acceptable excipient can be provided.

[0116] As another example of the present invention, a vaccine containing an immunogenic composition can be provided.

[0117] As another example of the present invention, there can be provided a method for producing an immunogenic conjugate of Streptococcus pneumoniae serotype 17F, comprising

[0118] (a) a step of fermenting and dissolving a bacterial cell producing capsular polysaccharide of Streptococcus pneumoniae serotype 17F;

[0119] (b) a step of purifying the capsular polysaccharide of serotype 17F Streptococcus pneumoniae in the dissolved cell;

[0120] (c) a step of hydrolyzing the purified capsular polysaccharide of Streptococcus pneumoniae serotype 17F to give the polysaccharide a desired size;

[0121] (d) a step of reacting the polysaccharide of the desired size obtained in the step (c) to activate the polysaccharide; and

[0122] (e) a step of combining the activated polysaccharide with a carrier protein to obtain a conjugate of Streptococcus pneumoniae serotype 17F capsular polysaccharide bound to a carrier protein.

[0123] A method for producing an immunogenic conjugate of Streptococcus pneumoniae serotype 17F may be provided, characterized in that the carrier protein used for combining in step (e) can form a conjugate with an activated polysaccharide by bringing it into interaction with a reducing agent.

[0124] Step (d) may include a reaction process using 0.01 ~ 0.22 μg of periodate per 1 μg of polysaccharide at a temperature of 20 to 25°C for a period of 15 to 20 hours.

[0125] The activated polysaccharide combined with the carrier protein in step (e) may have a molecular weight of 400 to 900 kDa.

[0126] The immunogenic conjugate may have a molecular weight of 300 to 4500 kDa.

[0127] The initial ratio of the administered activated serotype 17F capsular polysaccharide to the carrier protein (carrier protein:polysaccharide) can be 1:1.

[0128] In one example, at least 15 to 60% of the immunogenic conjugate may be present within 0.3 Kd of the molecular weight distribution when using a CL-4B column.

[0129] As one example of the present invention, an immunogenic conjugate obtained by the said method can be provided. As one example of the present invention, an immunogenic composition comprising the immunogenic conjugate obtained by the said method and a physiologically acceptable excipient can be provided. As another example, a vaccine comprising the immunogenic composition can be provided.

[0130] As one example of the present invention, there is provided an immunogenic conjugate of serotype 20 Streptococcus pneumoniae, comprising a capsular polysaccharide of serotype 20 isolated from Streptococcus pneumoniae; and a carrier protein associated with the capsular polysaccharide.

[0131] Serotype 20 polysaccharide with a molecular weight of 400 to 800 kDa can be activated and bind a carrier protein to form a conjugate.

[0132] In one example of the present invention, the immunogenic conjugate may have a molecular weight of 1000 to 4000 kDa, and, for example, the carrier protein may be a CRM 197 .

[0133] In one example of the present invention, the ratio of serotype 20 capsular polysaccharide to carrier protein in the immunogenic conjugate (w / w) may be from 1 to 5.

[0134] 70 to 90% of the serotype 20 immunogenic conjugate can be within 0.3 Kd using a CL-4B column. Another example provides an immunogenic conjugate of serotype 20 Streptococcus pneumoniae having an oxidation state of 4 to 16.

[0135] If the polysaccharide of Streptococcus pneumoniae serotype 20 is oxidized by adding 0.01 to 0.04 μg of periodate per 1 μg of sugar and conjugated with a protein, the molecular weight of the conjugate may be 1000 to 4000 kDa, and 70~90% of the conjugate may be within the specified range (0.3 kDa or less) of the molecular weight distribution, and the polysaccharide / protein ratio may be 1 to 5.

[0136] As one example of the present invention, an immunogenic composition comprising an immunogenic conjugate and a physiologically acceptable excipient can be provided.

[0137] As another example of the present invention, a vaccine containing an immunogenic composition can be provided.

[0138] As another example of the present invention, there can be provided a method for producing an immunogenic conjugate of Streptococcus pneumoniae serotype 20, comprising

[0139] (a) a step of fermenting and dissolving a bacterial cell producing capsular polysaccharide of serotype 20 Streptococcus pneumoniae;

[0140] (b) a step of purifying the capsular polysaccharide of serotype 20 Streptococcus pneumoniae in the dissolved cell;

[0141] (c) a step of bringing the polysaccharide into contact with an oxidizing agent to activate it; and

[0142] (d) a step of combining the activated polysaccharide with a carrier protein to obtain a conjugate of Streptococcus pneumoniae serotype 20 capsular polysaccharide bound to a carrier protein.

[0143] The carrier protein used for the combination in step (d) can be reacted with a reducing agent to obtain a conjugate with an activated polysaccharide.

[0144] Step (c) may comprise a reaction process using 0.01~0.04 μg of periodate per 1 μg of polysaccharide at a temperature of 20 to 25°C for a period of 15 to 20 hours.

[0145] The activated polysaccharide combined with the carrier protein in step (d) may have a molecular weight of 400 to 800 kDa.

[0146] In one example of the present invention, the immunogenic conjugate according to the present invention obtained by said method may have a molecular weight of 1000 to 4000 kDa.

[0147] The initial ratio of the administered activated serotype 20 capsular polysaccharide to the carrier protein (carrier protein:polysaccharide) can be 1:1.

[0148] In one example, at least 70 to 90% of the immunogenic conjugate may be within 0.3 Kd of the molecular weight distribution when using a CL-4B column.

[0149] As one example of the present invention, an immunogenic conjugate obtained by the said method can be provided.

[0150] As one example, an immunogenic composition may be provided comprising an immunogenic conjugate obtained by the said method and a physiologically acceptable filler.

[0151] As another example, a vaccine containing an immunogenic composition may be provided.

[0152] In one embodiment of the present invention, an immunogenic composition is provided, comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, comprising a capsular polysaccharide isolated from one or more serotypes selected from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F; and a carrier protein conjugated with the corresponding capsular polysaccharides.

[0153] In one embodiment, an immunogenic composition is provided comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein if the immunogenic composition comprises a polysaccharide isolated from serotype 2, the polysaccharide having a molecular weight of 100 to 400 kDa is activated and binds the carrier protein to form a conjugate, or

[0154] if the immunogenic composition contains a polysaccharide isolated from serotype 17F, then the polysaccharide with a molecular weight of 400 to 900 kDa is activated and binds the carrier protein to form a conjugate, or

[0155] If the immunogenic composition contains a polysaccharide isolated from serotype 20, the polysaccharide with a molecular weight of 400 to 800 kDa is activated and binds the carrier protein to form a conjugate.

[0156] In one embodiment of the present invention, there is provided an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein if the immunogenic composition comprises a polysaccharide isolated from serotype 2, then the immunogenic conjugate comprising a polysaccharide isolated from serotype 2 has a molecular weight of 1,000 to 16,000 kDa, or

[0157] if the immunogenic composition contains a polysaccharide isolated from serotype 17F, then the immunogenic conjugate containing the polysaccharide isolated from serotype 17F has a molecular weight of 300 to 4500 kDa, or

[0158] if the immunogenic composition contains a polysaccharide isolated from serotype 20, then the immunogenic conjugate containing the polysaccharide isolated from serotype 20 has a molecular weight of 1000 to 4000 kDa.

[0159] In one embodiment of the present invention, there is provided an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, characterized in that the carrier protein is TT (tetanus toxoid) or CRM 197 , and preferably the immunogenic composition containing only one serotype may contain CRM 197 as a carrier protein.

[0160] In one embodiment of the present invention, there is provided an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein if the immunogenic composition comprises a polysaccharide isolated from serotype 2, then the ratio of serotype 2 capsular polysaccharide to carrier protein in the immunogenic conjugate (polysaccharide / protein, (w / w)) is from 0.5 to 2.0, or

[0161] if the immunogenic composition contains a polysaccharide isolated from serotype 17F, then the ratio of the capsular polysaccharide of serotype 17F to the carrier protein in the immunogenic conjugate (polysaccharide / protein, (w / w)) is from 0.5 to 18, or

[0162] If the immunogenic composition contains a polysaccharide isolated from serotype 20, the ratio of the capsular polysaccharide of serotype 20 to the carrier protein in the immunogenic conjugate (polysaccharide / protein, (w / w)) is from 1 to 5.

[0163] In one embodiment of the present invention, there is provided an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein in the molecular weight distribution from 20 to 60% of the conjugate is within 0.3 Kd when using a CL-4B column in the case of an immunogenic conjugate comprising a polysaccharide isolated from serotype 2, or

[0164] in a molecular weight distribution of 15 to 60% of the conjugate is within 0.3 Kd when using a CL-4B column in the case of an immunogenic conjugate containing a polysaccharide isolated from serotype 17F, or

[0165] In the molecular weight distribution of 70 to 90% of the conjugate, it is within 0.3 Kd when using the CL-4B column in the case of the immunogenic conjugate containing the polysaccharide isolated from serotype 20.

[0166] In one embodiment of the present invention, there is provided an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein

[0167] the oxidation state of the conjugated polysaccharide in the conjugate is from 2 to 18 in the case of an immunogenic conjugate containing a polysaccharide isolated from serotype 2, or

[0168] the oxidation state of the conjugated polysaccharide in the conjugate is from 1 to 22 in the case of an immunogenic conjugate containing a polysaccharide isolated from serotype 17F, or

[0169] The oxidation state of the conjugated polysaccharide in the conjugate is from 4 to 16 in the case of an immunogenic conjugate containing a polysaccharide isolated from serotype 20.

[0170] In one embodiment of the present invention, there is provided an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein the immunogenic composition is a composition in which polysaccharides isolated from 15 serotypes distinct from each other are conjugated to corresponding carrier proteins,

[0171] and serotypes include 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F and 23F, and

[0172] serotypes are conjugated with CRM 197.

[0173] In one embodiment of the present invention, there is provided an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein the immunogenic composition is a composition in which polysaccharides isolated from 23 serotypes distinct from each other are conjugated to corresponding carrier proteins,

[0174] and serotypes include 1, 2, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F,

[0175] and among these serotypes, capsular polysaccharides isolated from serotypes 3 and 5 are conjugated to the TT carrier protein, and capsular polysaccharides isolated from serotypes 1, 2, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F are conjugated to the CRM197 carrier protein.

[0176] In one embodiment of the present invention, there is provided an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein the immunogenic composition is a composition in which polysaccharides isolated from 24 serotypes distinct from each other are conjugated to corresponding carrier proteins,

[0177] and serotypes include 2, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F,

[0178] and among these serotypes, capsular polysaccharides isolated from serotypes 1 and 5 are conjugated to the TT carrier protein, and capsular polysaccharides isolated from serotypes 2, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F are conjugated to the CRM197 carrier protein.

[0179] In one embodiment of the present invention, an immunogenic composition is provided comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein the immunogenic composition comprises a physiologically acceptable filler.

[0180] In one embodiment of the present invention, an immunogenic composition is provided comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, characterized in that said immunogenic composition is a vaccine.

[0181] In one embodiment of the present invention, a method for producing an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate comprising

[0182] (a) a step of fermenting and dissolving a bacterial cell producing a capsular polysaccharide isolated from one or more serotypes selected from the group consisting of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F;

[0183] (b) a step of purifying the capsular polysaccharide isolated from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F in the dissolved cell;

[0184] (c) a step of bringing the purified polysaccharide into contact with an oxidizing agent to activate it; and

[0185] (d) a step of combining the activated polysaccharide with a carrier protein to obtain a Streptococcus pneumoniae polysaccharide-protein conjugate linked to a carrier protein.

[0186] In one embodiment of the present invention, the production method may further comprise a step of hydrolyzing the purified capsular polysaccharide of Streptococcus pneumoniae to give the polysaccharide a desired size before step (c) in the case of capsular polysaccharides isolated from serotypes 2 and 17F.

[0187] In one embodiment of the present invention, a method for producing an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate is provided, characterized in that the carrier protein used for combining in step (d) forms a conjugate with an activated polysaccharide by reacting with one or more reducing agents selected from the group consisting of cyanoborohydride, borane-pyridine and a borohydride ion exchange resin.

[0188] In one embodiment of the present invention, a method for producing an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate is provided, characterized in that step (c) is reacted using 0.01 ~ 0.22 μg of periodate per 1 μg of polysaccharide at a temperature of 20 to 25°C for a period of 15 to 20 hours.

[0189] In one embodiment of the present invention, there is provided a Streptococcus pneumoniae polysaccharide-protein conjugate for preventing or treating Streptococcus pneumoniae infection, obtained by the said method.

[0190] As one embodiment, a method for preventing or treating an infection caused by Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and / or 33F in a subject may be provided,

[0191] by administering an effective dose of an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate containing

[0192] a capsular polysaccharide isolated from one or more serotypes of Streptococcus pneumoniae selected from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F; and

[0193] one or 2 or more carrier proteins conjugated to corresponding capsular polysaccharides. "And / or" means "and" or "or".

[0194] The method can prevent or treat an infection caused by one or more serotypes selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, or infection with one or more serotypes can occur, and the method can prevent infection with 15 serotypes, or the method can prevent infection with 23 serotypes, or the method can prevent infection with 24 serotypes.

[0195] In one example of the present invention, for the prevention or treatment of pneumococcal infection, there is provided the use of a Streptococcus pneumoniae polysaccharide-protein conjugate containing

[0196] a capsular polysaccharide isolated from one or more serotypes of Streptococcus pneumoniae selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F; and

[0197] one or 2 or more carrier proteins conjugated to corresponding capsular polysaccharides.

[0198] In one example of the present invention, there is provided an immunogenic conjugate of Streptococcus pneumoniae serotype, comprising a capsular polysaccharide of serotype 2 isolated from Streptococcus pneumoniae; and a carrier protein associated with the capsular polysaccharide.

[0199] Serotype 2 polysaccharide with a molecular weight of 100 to 400 kDa can be activated and bind a carrier protein to form a conjugate.

[0200] In one example of the present invention, the immunogenic conjugate may have a molecular weight of 1,000 to 16,000 kDa, and, for example, the carrier protein may be a CRM 197 .

[0201] In one example of the present invention, the ratio of serotype 2 capsular polysaccharide to carrier protein in the immunogenic conjugate (w / w) may be from 0.5 to 2.0.

[0202] 20 to 60% of the serotype 2 immunogenic conjugate can be within 0.3 Kd using the CL-4B column. Another example provides an immunogenic conjugate of Streptococcus pneumoniae serotype 2 having an oxidation state of 2 to 18.

[0203] If the polysaccharide of Streptococcus pneumoniae serotype 2 is oxidized by adding 0.02 to 0.12 μg of periodate per 1 μg of sugar and conjugated with a protein, the molecular weight of the conjugate may be 1000 ~ 16000 kDa, and 20 ~ 60% of the conjugate may be within the specified range (0.3 kd or less) of the molecular weight distribution, and the polysaccharide / protein ratio may be 0.5 to 2.0.

[0204] As one example of the present invention, an immunogenic composition comprising an immunogenic conjugate and a physiologically acceptable excipient can be provided.

[0205] As another example of the present invention, a vaccine containing an immunogenic composition can be provided.

[0206] As another example of the present invention, a vaccine containing an immunogenic composition can be provided.

[0207] As another example of the present invention, there can be provided a method for producing an immunogenic conjugate of serotype 2 Streptococcus pneumoniae, comprising

[0208] (a) a step of fermenting and dissolving a bacterial cell producing capsular polysaccharide of serotype 2 Streptococcus pneumoniae;

[0209] (b) a step of purifying the capsular polysaccharide of serotype 2 Streptococcus pneumoniae in the dissolved cell;

[0210] (c) a step of hydrolyzing the purified capsular polysaccharide of serotype 2 Streptococcus pneumoniae to give the polysaccharide a desired size;

[0211] (d) a step of bringing the polysaccharide of the desired size obtained in the step (c) into reaction to activate the polysaccharide; and

[0212] (e) a step of combining the activated polysaccharide with a carrier protein to obtain a carrier protein-linked Streptococcus pneumoniae serotype 2 capsular polysaccharide conjugate.

[0213] The carrier protein used for the combination in step (e) can form a conjugate with the activated polysaccharide by reacting with a reducing agent.

[0214] Step (d) may include a reaction process using 0.02~0.12 μg of periodate per 1 μg of polysaccharide at a temperature of 20 to 25°C for a period of 15 to 20 hours.

[0215] The activated polysaccharide combined with the carrier protein in step (e) may have a molecular weight of 100 to 400 kDa.

[0216] A method for producing an immunogenic conjugate of Streptococcus pneumoniae serotype 2, wherein the carrier protein is CRM197, may be provided.

[0217] The immunogenic conjugate may have a molecular weight of 1000 to 16000 kDa.

[0218] In one example, the initial ratio of the administered activated serotype 2 capsular polysaccharide to the carrier protein (carrier protein:polysaccharide) may be from 0.5 to 2:1.

[0219] As another example, a method for producing an immunogenic conjugate of Streptococcus pneumoniae serotype 2 can be provided, wherein at least 20 to 60% of the immunogenic conjugate is within 0.3 Kd when using a CL-4B column.

[0220] As one example of the present invention, an immunogenic conjugate obtained by the said method can be provided.

[0221] As one example of the present invention, an immunogenic composition may be provided comprising an immunogenic conjugate obtained by the said method and a physiologically acceptable filler.

[0222] As another example, a vaccine containing an immunogenic composition may be provided.

[0223] The present invention provides an immunogenic conjugate of serotype 9N Streptococcus pneumoniae, comprising a capsular polysaccharide of serotype 9N isolated from Streptococcus pneumoniae; and a carrier protein associated with the capsular polysaccharide.

[0224] In one example of the present invention, a serotype 9N polysaccharide with a molecular weight of 200 to 700 kDa can be activated and bind a carrier protein to form a conjugate.

[0225] The immunogenic conjugate may have a molecular weight of 500 to 4000 kDa, and the carrier protein may be a CRM 197 .

[0226] The ratio of serotype 9N capsular polysaccharide to the carrier protein in the immunogenic conjugate (w / w) may be from 0.1 to 5, and may preferably be 0.5 ~ 2.5.

[0227] In one example, 15 to 60% of the immunogenic conjugate can be within 0.3 Kd when using a CL-4B column.

[0228] In one example, the conjugate may have an oxidation state of from 2 to 19.

[0229] In one example of the present invention, if the polysaccharide of Streptococcus pneumoniae serotype 9N is oxidized by adding 0.02~0.19 μg of periodate per 1 μg of sugar and conjugated with a protein, the molecular weight of the conjugate can be 500~4000 kDa, and 15~60% of the conjugate can be within a predetermined range (0.3 kd or less) of the molecular weight distribution, and the polysaccharide / protein ratio can be 0.5~2.5.

[0230] As one example of the present invention, an immunogenic composition comprising an immunogenic conjugate and a physiologically acceptable excipient can be provided.

[0231] As another example of the present invention, a vaccine containing an immunogenic composition can be provided.

[0232] As another example of the present invention, there may be provided a method for producing an immunogenic conjugate of serotype 9N Streptococcus pneumoniae, comprising

[0233] (a) a step of fermenting and dissolving a bacterial cell producing capsular polysaccharide of serotype 9N Streptococcus pneumoniae;

[0234] (b) a step of purifying the capsular polysaccharide of serotype 9N Streptococcus pneumoniae in the dissolved cell;

[0235] (c) a step of bringing the polysaccharide into contact with an oxidizing agent to activate it; and

[0236] (d) a step of combining the activated polysaccharide with a carrier protein to obtain a conjugate of Streptococcus pneumoniae serotype 9N capsular polysaccharide bound to a carrier protein.

[0237] A method for producing an immunogenic conjugate of serotype 9N Streptococcus pneumoniae may be provided, characterized in that the carrier protein used for combining in step (d) is brought into contact with a reducing agent to produce a conjugate with an activated polysaccharide.

[0238] Step (c) may comprise a reaction process using 0.02 ~ 0.19 μg of periodate per 1 μg of polysaccharide at a temperature of 20 to 25°C for a period of 15 to 20 hours.

[0239] The polysaccharide that reacts with the oxidizing agent in step (c) may have a molecular weight of 400 to 900 kDa.

[0240] The activated polysaccharide combined with the carrier protein in step (d) may have a molecular weight of 200-700 kDa.

[0241] The immunogenic conjugate may have a molecular weight of 500 to 4000 kDa.

[0242] The initial ratio of the administered activated serotype 9N capsular polysaccharide to the carrier protein (carrier protein:polysaccharide) can be from 0.5 to 2.5:1.

[0243] In one example, at least 15% to 60% of the immunogenic conjugate may be within 0.3 Kd when using a CL-4B column.

[0244] As one example of the present invention, an immunogenic conjugate obtained by the said method can be provided. As one example of the present invention, an immunogenic composition comprising the immunogenic conjugate obtained by the said method and a physiologically acceptable excipient can be provided. As another example, a vaccine comprising the immunogenic composition can be provided.

[0245] In one example of the present invention, there is provided an immunogenic conjugate of serotype 17F Streptococcus pneumoniae comprising a capsular polysaccharide of serotype 17F isolated from Streptococcus pneumoniae; and a carrier protein associated with the capsular polysaccharide.

[0246] Serotype 17F polysaccharide with a molecular weight of 400 to 900 kDa can be activated and bind a carrier protein to form a conjugate.

[0247] In one example of the present invention, the immunogenic conjugate may have a molecular weight of 300 to 4500 kDa, and, for example, the carrier protein may be a CRM 197 .

[0248] In one example of the present invention, the ratio of serotype 17F capsular polysaccharide to carrier protein in the immunogenic conjugate (w / w) may be from 0.5 to 18.

[0249] From 15% to 60% of the immunogenic conjugate of serotype 17F can be detected within 0.3 Kd using the CL-4B column. Another example provides an immunogenic conjugate of serotype 17F Streptococcus pneumoniae having an oxidation state of 1 to 22.

[0250] If the polysaccharide of Streptococcus pneumoniae serotype 17F is oxidized by adding 0.01 to 0.22 μg of periodate per 1 μg of sugar and conjugated with a protein, the molecular weight of the conjugate may be 300 to 4500 kDa, and 15~60% of the conjugate may be within the given range (0.3 kd or less) of the molecular weight distribution, and the polysaccharide / protein ratio may be 0.5 to 18.

[0251] As one example of the present invention, an immunogenic composition comprising an immunogenic conjugate and a physiologically acceptable excipient can be provided.

[0252] As another example of the present invention, a vaccine containing an immunogenic composition can be provided.

[0253] As another example of the present invention, there can be provided a method for producing an immunogenic conjugate of Streptococcus pneumoniae serotype 17F, comprising

[0254] (a) a step of fermenting and dissolving a bacterial cell producing capsular polysaccharide of Streptococcus pneumoniae serotype 17F;

[0255] (b) a step of purifying the capsular polysaccharide of serotype 17F Streptococcus pneumoniae in the dissolved cell;

[0256] (c) a step of hydrolyzing the purified capsular polysaccharide of Streptococcus pneumoniae serotype 17F to give the polysaccharide a desired size;

[0257] (d) a step of bringing the polysaccharide of the desired size obtained in step (c) into reaction to activate the polysaccharide; and

[0258] (e) a step of combining the activated polysaccharide with a carrier protein to obtain a conjugate of Streptococcus pneumoniae serotype 17F capsular polysaccharide bound to a carrier protein.

[0259] A method for producing an immunogenic conjugate of Streptococcus pneumoniae serotype 17F can be provided, characterized in that the carrier protein used for combining in step (e) can form a conjugate with an activated polysaccharide by reacting with a reducing agent.

[0260] Step (d) may include a reaction process using 0.01 ~ 0.22 μg of periodate per 1 μg of polysaccharide at a temperature of 20 to 25°C for a period of 15 to 20 hours.

[0261] The activated polysaccharide combined with the carrier protein in step (e) may have a molecular weight of 400 to 900 kDa.

[0262] The immunogenic conjugate may have a molecular weight of 300 to 4500 kDa.

[0263] The initial ratio of the administered activated capsular polysaccharide of serotype 17F to the carrier protein (carrier protein:polysaccharide) can be 1:1.

[0264] In one example, at least 15% to 60% of the immunogenic conjugate may be within 0.3 Kd of the molecular weight distribution when using a CL-4B column.

[0265] As one example of the present invention, an immunogenic conjugate obtained by the said method can be provided. As one example of the present invention, an immunogenic composition comprising the immunogenic conjugate obtained by the said method and a physiologically acceptable excipient can be provided. As another example, a vaccine comprising the immunogenic composition can be provided.

[0266] In one example of the present invention, there is provided an immunogenic conjugate of serotype 20 Streptococcus pneumoniae comprising a capsular polysaccharide of serotype 20 isolated from Streptococcus pneumoniae; and a carrier protein associated with the capsular polysaccharide.

[0267] Serotype 20 polysaccharide with a molecular weight of 400 to 800 kDa can be activated and bind a carrier protein to form a conjugate.

[0268] In one example of the present invention, the immunogenic conjugate may have a molecular weight of 1000 to 4000 kDa, and, for example, the carrier protein may be a CRM 197 .

[0269] In one example of the present invention, the ratio of serotype 20 capsular polysaccharide to carrier protein in the immunogenic conjugate (w / w) may be from 1 to 5.

[0270] 70 to 90% of the serotype 20 immunogenic conjugate can be within 0.3 Kd using a CL-4B column. Another example is a Streptococcus pneumoniae serotype 20 immunogenic conjugate having an oxidation state of 4 to 16.

[0271] If the polysaccharide of Streptococcus pneumoniae serotype 20 is oxidized by adding 0.01 to 0.04 μg of periodate per 1 μg of sugar and conjugated with a protein, the molecular weight of the conjugate may be 1000 to 4000 kDa, and 70~90% of the conjugate may be within a given range (0.3 kDa or less) of the molecular weight distribution, and the polysaccharide / protein ratio may be 1 to 5.

[0272] As one example of the present invention, an immunogenic composition comprising an immunogenic conjugate and a physiologically acceptable excipient can be provided.

[0273] As another example of the present invention, a vaccine containing an immunogenic composition can be provided.

[0274] As another example of the present invention, there can be provided a method for producing an immunogenic conjugate of Streptococcus pneumoniae serotype 20, comprising

[0275] (a) a step of fermenting and dissolving a bacterial cell producing capsular polysaccharide of serotype 20 Streptococcus pneumoniae;

[0276] (b) a step of purifying the capsular polysaccharide of serotype 20 Streptococcus pneumoniae in the dissolved cell;

[0277] (c) a step of bringing the polysaccharide into contact with an oxidizing agent to activate it; and

[0278] (d) a step of combining the activated polysaccharide with a carrier protein to obtain a conjugate of Streptococcus pneumoniae serotype 20 capsular polysaccharide bound to a carrier protein.

[0279] The carrier protein used for the combination in step (d) can be reacted with a reducing agent to obtain a conjugate with an activated polysaccharide.

[0280] Step (c) may comprise a reaction process using 0.01 ~ 0.04 μg of periodate per 1 μg of polysaccharide at a temperature of 20 to 25°C for a period of 15 to 20 hours.

[0281] The activated polysaccharide combined with the carrier protein in step (d) may have a molecular weight of 400 to 800 kDa.

[0282] In one example of the present invention, the immunogenic conjugate according to the present invention obtained by said method may have a molecular weight of 1000 to 4000 kDa.

[0283] The initial ratio of the administered activated serotype 20 capsular polysaccharide to the carrier protein (carrier protein:polysaccharide) can be 1:1.

[0284] In one example, at least 70 to 90% of the immunogenic conjugate may be within 0.3 Kd of the molecular weight distribution when using a CL-4B column.

[0285] As one example of the present invention, an immunogenic conjugate obtained by the said method can be provided.

[0286] As one example, an immunogenic composition may be provided comprising an immunogenic conjugate obtained by the said method and a physiologically acceptable filler.

[0287] As another example, a vaccine containing an immunogenic composition may be provided.

[0288] Characteristics of serotype 20 polysaccharide-carrier protein conjugate

[0289] In one embodiment, the conjugate may have a molecular weight of 1300 to 4300 kDa, or a molecular weight of 1250 to 4250 kDa, or a molecular weight of 1200 to 4200 kDa, or a molecular weight of 1150 to 4150 kDa, or a molecular weight of 1100 to 4100 kDa, or a molecular weight of 1000 to 4000 kDa. All numerical values ​​included in any of the above ranges are considered as a separate embodiment.

[0290] For the above molecular weight ranges, the conjugate can be obtained with consistently excellent yield. Furthermore, the relative free sugar content can be reduced. Furthermore, the above molecular weight ranges can be associated with excellent immunogenicity.

[0291] The immunogenic composition according to the present invention is obtained by combining the corresponding polysaccharide-protein conjugates after purification thereof.

[0292] The serotype polysaccharide-protein conjugate according to the present invention can be characterized by the ratio of saccharide to carrier protein (amount of polysaccharide / amount of protein) (w / w).

[0293] In some embodiments, the ratio of saccharide to carrier protein for each serotype in the polysaccharide-protein conjugate (w / w) can be from 0.1 to 7, 0.2 to 7.5, 0.3 to 7, 0.4 to 6.5, 0.5 to 6, 0.6 to 6.5, 0.7 to 6, 0.8 to 5.8, 0.9 to 5.6, 0.95 to 5.3, or 1 to 5. For example, it can be about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.

[0294] In another embodiment, the ratio of saccharide to carrier protein (w / w) may be from 1 to 5, from 1.2 to 4.5, or from 1.3 to 4.

[0295] Preferably, the carrier protein may be CRM197.

[0296] If the saccharide to carrier protein ratio is as described above, the conjugate can be obtained with consistently excellent yield. Furthermore, the relative free sugar content can be reduced. Furthermore, within the ranges given above, immunogenicity is excellent, and the conjugate can be maintained in a stable form without adverse effects from other serotypes.

[0297] The conjugate and immunogenic composition according to the present invention may comprise a free saccharide that is not conjugated to the carrier protein via a covalent bond, but is present in the polysaccharide-protein conjugate composition. The free saccharide may be associated with the polysaccharide-protein conjugate by non-covalent bonds (i.e., it may be non-covalently bonded or adsorbed in the polysaccharide-protein conjugate, or may be encapsulated in the polysaccharide-protein conjugate or the polysaccharide-protein conjugate).

[0298] In a preferred embodiment, the polysaccharide-protein conjugate comprises free polysaccharide of each serotype in an amount of less than about 70%, about 60%, about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10% of the total amount of polysaccharide of each type.

[0299] The polysaccharide-protein conjugate of each serotype can also be characterized by its molecular weight distribution (Kd). The relative size distribution of the conjugate molecules can be measured using a size-exclusion chromatography medium (CL-4B, cross-linked agarose beads, 4%). The molecular weight distribution profile of the conjugate is obtained by size-exclusion chromatography (SEC) on a gravity column. Large molecules excluded from the pores of the medium elute faster than small molecules. The column eluates are collected using a fraction collector. Colorimetric analysis of saccharides in the fractions is performed. To measure Kd, the column is calibrated to determine the fractions whose molecules are completely excluded from the pores (V0), (Kd = 0), and the fractions for which maximum retention is observed (Vi), (Kd = 1). The fractions in which the specified sample characteristic (Ve) is achieved are related to Kd by the equation Kd = (Ve - V0) / (Vi - V0).

[0300] In a preferred embodiment, at least 30% of the polysaccharide-protein conjugate of each serotype can be within 0.3 Kd when using a CL-4B column.

[0301] In a preferred embodiment, at least 95% of the polysaccharide-protein conjugate of each serotype can be within 0.3 Kd when using a CL-4B column. In a preferred embodiment, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the polysaccharide-protein conjugate of each serotype can be within 0.3 Kd when using a CL-4B column. In a preferred embodiment, at least 60% of the polysaccharide-protein conjugate of each serotype can be within 0.3 Kd when using a CL-4B column. In a preferred embodiment, 50 to 90% of the polysaccharide-protein conjugate of each serotype can be within 0.3 Kd when using a CL-4B column. In a preferred embodiment, 65 to 90% of the polysaccharide-protein conjugate of each serotype can be within 0.3 Kd when using a CL-4B column.In a preferred embodiment, 70 to 90% of the polysaccharide-protein conjugate of each serotype may be within 0.3 Kd when using a CL-4B column.

[0302] 1.4 Capsule saccharide-carrier protein combination

[0303] In one embodiment, the immunogenic composition of the present invention comprises a conjugate of one or more polysaccharides isolated from the group of Streptococcus pneumoniae serotypes consisting of serotypes 2, 9N, 17F and 20 with a protein. In one embodiment, any of the immunogenic compositions may comprise a conjugate in which the polysaccharide isolated from one or more serotypes selected from the group consisting of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F is further conjugated to a carrier protein.

[0304] In one embodiment, any polysaccharide-protein conjugates in the immunogenic composition are conjugated to CRM197 and / or TT, respectively. Preferably, the 23-valent or 24-valent immunogenic composition may simultaneously contain CRM197 and TT as carrier proteins, in which case serotype 5 preferably contains TT as the carrier protein. In one example, in a 23-valent immunogenic composition, polysaccharides isolated from serotypes 3 and 5 are conjugated to TT, and polysaccharides isolated from serotypes 1, 2, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F are conjugated to CRM197. In one example, in a 24-valent immunogenic composition, polysaccharides isolated from serotypes 1 and 5 are conjugated to TT, and serotypes 2, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F are conjugated to CRM197.

[0305] In one embodiment, the immunogenic composition may comprise a polysaccharide-protein conjugate isolated from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 different serotypes.

[0306] 2. Dose of immunogenic composition

[0307] The amount of capsular saccharide-carrier protein conjugate(s) in each dose is selected to induce a protective immune response without the significant side effects of conventional vaccines. The amount specified above can be varied depending on the specific immunogen being used and the method of delivery.

[0308] 2.1 Quantity of capsular polysaccharide-carrier protein conjugate

[0309] The amount of a specific capsular polysaccharide-carrier protein conjugate in an immunogenic composition can be calculated based on the ratio of the total amount of polysaccharides (conjugated and unconjugated) to the conjugate. For example, if the capsular polysaccharide-carrier protein conjugate contains 20% free polysaccharides, this means that a 100 μg dose of polysaccharide contains approximately 80 μg of conjugated polysaccharides and approximately 20 μg of unconjugated polysaccharides. The amount of polysaccharide-protein conjugate may vary depending on the pneumococcal serotype. The concentration of polysaccharide can be measured using an anthrone or uronic acid assay.

[0310] The "immunogenic amount" of the different polysaccharide components in the immunogenic composition may be different, and the composition may contain any specific polysaccharide antigen in an amount of about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 15 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg or about 100 μg, respectively.

[0311] In general, each dose may contain from 0.1 μg to 100 μg, in particular from 0.5 μg to 20 μg, more specifically from 1.0 μg to 10 μg, and more specifically from 2.0 μg to 5.0 μg of polysaccharides of a given serotype. All numerical values ​​included in the above ranges are considered as a separate embodiment.

[0312] In one embodiment, each dose may contain polysaccharides in an amount of about 1.0 μg, about 1.2 μg, about 1.4 μg, about 1.6 μg, about 1.8 μg, about 2.0 μg, about 2.2 μg, about 2.4 μg, about 2.6 μg, about 2.8 μg, about 3.0 μg, about 3.2 μg, about 3.4 μg, about 3.6 μg, about 3.8 μg, about 4.0 μg, about 4.2 μg, about 4.4 μg, about 4.6 μg, about 4.8 μg, about 5.0 μg, about 5.2 μg, about 5.4 μg, about 5.6 μg, approximately 5.8 μg or approximately 6.0 μg in each particular capsular saccharide-carrier protein conjugate.

[0313] In one embodiment, each dose may contain polysaccharides in an amount of 1 to 3 μg in polysaccharide-protein conjugates isolated from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and / or 33F. For example, a dose may contain polysaccharides in an amount of about 1.1 mcg, about 1.2 mcg, about 1.3 mcg, about 1.4 mcg, about 1.5 mcg, about 1.6 mcg, about 1.7 mcg, about 1.8 mcg, about 1.9 mcg, about 2.0 mcg, about 2.1 mcg, about 2.2 mcg, about 2.3 mcg, about 2.4 mcg, about 2.5 mcg, about 2.6 mcg, about 2.7 mcg, about 2.8 mcg, about 2.9 mcg, or about 3.0 mcg.

[0314] In one embodiment, if a polysaccharide-protein conjugate isolated from serotype 6B is additionally contained, then from 2 to 6 μg of polysaccharides may be contained.

[0315] 2.2 Amount of carrier protein

[0316] In one embodiment, the carrier protein may be CRM197 and / or TT.

[0317] In one embodiment, if the carrier protein is CRM197, then each dose of the immunogenic composition may contain carrier proteins in an amount of 10 μg to 150 μg, 20 μg to 100 μg, or 25 μg to 95 μg. For example, a 24-valent immunogenic composition according to one embodiment of the present invention may contain 70 to 90 μg of carrier proteins.

[0318] In one embodiment, if the carrier protein is TT, then each dose of the immunogenic composition may contain carrier proteins in an amount of 5 μg to 15 μg, 8 μg to 10 μg.

[0319] 3. Adjuvant

[0320] In some embodiments, the immunogenic composition described in the present invention may further comprise one or more adjuvants. The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An adjuvant may enhance the immune response to an antigen that has low immunogenicity, and / or may increase the antibody titer to the antigen, and / or may reduce the effective dose of the antigen to achieve an immune response in a subject if, in the case of a single administration, an increase in the antibody titer does not occur or is weakly expressed or a cellular immune response is not induced. Thus, the effect of an adjuvant is mainly to enhance the immune response, which is known to those skilled in the art. Suitable adjuvants that enhance the effectiveness of the composition include, but are not limited to, the following substances:

[0321] In one example, the adjuvant may include aluminum (alum.) salts, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.

[0322] In a specific embodiment, the adjuvant is an aluminum salt. The aluminum salt adjuvant may be an alum-precipitated vaccine or an alum-adsorbed vaccine. Aluminum salt adjuvants are known in the art. Aluminum salts include, but are not limited to, hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, alhydrogel, superphos, amphogel, aluminum (III) hydroxide, aluminum hydroxyphosphate sulfate (aluminum phosphate adjuvant (APA)), amorphous alumina, trihydrated alumina, or trihydroxyaluminum.

[0323] APA is an aqueous suspension of aluminum hydroxyphosphate. APA is produced by mixing aluminum chloride and sodium phosphate at a 1:1 volume ratio and precipitating the aluminum hydroxyphosphate. After mixing, the target aggregate particles, ranging in size from 2 to 8 μm, are obtained by grinding the material in a high-shear mixer. The products are then diafiltered in saline and sterilized with steam.

[0324] In a specific embodiment, the protein is adsorbed at a ratio of 50-200 g protein / mg aluminum hydroxide using commercially available Al(OH)3 (e.g., alhydrogel or superphos manufactured by Denmark / Accurate Chemical and Scientific Co. (US New York Westbury). In another embodiment, the level of protein adsorption may vary depending on the pI (isoelectric pH) of the protein and the pH of the medium. Proteins with a reduced pI are more actively adsorbed to positively charged aluminum ions compared to a protein with an increased pI. The aluminum salt can form an Al depot, providing a slow release over 2-3 weeks, and / or can participate in non-specific activation of macrophages and complement activation, and / or can stimulate the innate immune mechanism (possibly through stimulation with uric acid).

[0325] In a preferred embodiment, the adjuvant is an aluminum-based adjuvant selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide. In one embodiment, the immunogenic composition described herein comprises an aluminum phosphate-based adjuvant.

[0326] 4. Composition

[0327] The immunogenic composition according to the present invention can be prepared in liquid form (i.e., as a solution or suspension) or in lyophilized form. Preferably, the liquid formulation can be administered directly in the form in which it is packaged, and thus, this formulation is ideally suited for injection and does not require reconstitution in an aqueous medium, which is required in the case of the lyophilized composition according to the present invention.

[0328] The immunogenic composition according to the present invention can be prepared by a method approved in the art. For example, the composition can be prepared by combining a separate pneumococcal conjugate with a physiologically acceptable excipient. Examples of excipients such as those mentioned above include, but are not limited to, water, buffered saline, a polyhydric alcohol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and dextrose solution.

[0329] The present invention provides an immunogenic composition comprising any of the combinations of the described polysaccharide-protein conjugates and pharmaceutically acceptable excipients, carriers or diluents.

[0330] In one embodiment, the immunogenic composition according to the present invention is in liquid form, preferably an aqueous liquid form.

[0331] The immunogenic composition according to the present invention may contain one or more kinds of buffers, salts, divalent cations, nonionic detergents, cryoprotectants such as sugars, antioxidants such as free radical scavengers, and chelating agents, and any different combinations thereof.

[0332] In one embodiment, the immunogenic composition of the present invention comprises a buffer. In one embodiment, the buffer has a pKa of from about 3.5 to about 7.5. In some embodiments, the buffer is a phosphate, succinate, histidine, or citrate buffer. In some embodiments, the buffer comprises succinate at a final concentration of from 1 mM to 10 mM. In one specific embodiment, the final concentration of succinate is about 5 mM.

[0333] In one embodiment, the immunogenic composition of the present invention comprises a salt. In some embodiments, the salt is selected from the group consisting of magnesium chloride, potassium chloride, sodium chloride, and combinations thereof. In a preferred embodiment, the salt is sodium chloride. In one specific embodiment, the immunogenic composition of the present invention comprises 150 mM sodium chloride.

[0334] In one embodiment, the immunogenic composition according to the present invention comprises a surfactant. The surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid ester, polysorbate 80 (Tween 80), polysorbate 60 (Tween 60), polysorbate 40 (Tween 40) and polysorbate 20 (Tween 20), polyoxyethylene alkyl ether (including, but not limited to, Brij 58, Brij 35), and other materials, such as one or more kinds of non-ionic surfactants, including, but not limited to, Triton X-100; Triton X-114, NP40, Span 85 and Pluronic series non-ionic surfactants (e.g., Pluronic 121). In a preferred embodiment, the immunogenic composition comprises polysorbate-80 or polysorbate-20, preferably polysorbate-20.In a preferred embodiment, the immunogenic composition comprises polysorbate-20 at a concentration of from about 0.001% to about 2% (preferably less than about 0.005%).

[0335] In one embodiment, the container according to the present invention is made of glass, metal (e.g., steel, stainless steel, aluminum, etc.) and / or polymers (e.g., thermoplastic materials, elastomers, thermoplastic elastomers). In one embodiment, the container according to the present invention is made of glass.

[0336] In one embodiment, the present invention provides an injectable preparation filled with any of the immunogenic compositions described herein. In one embodiment, the injectable preparation is treated with silicone and / or is prepared in a glass container.

[0337] 5. Application

[0338] In one embodiment, the immunogenic composition described in the present invention is intended for use as a pharmaceutical. The amount of conjugate in the composition is selected to induce a protective immune response without significant side effects. This amount may vary depending on the pneumococcal serotype.

[0339] The immunogenic composition described in the present invention can be used in various therapeutic or prophylactic methods for the prevention, treatment, or amelioration of bacterial infections, diseases, or conditions in a subject. In particular, the immunogenic composition described in the present invention can be used to prevent, treat, or ameliorate Streptococcus pneumoniae infections, diseases, or conditions in a subject.

[0340] The contents of all materials or patent applications cited in the description of this patent application are incorporated herein by reference.

[0341] The present invention is illustrated in the accompanying examples. The following examples are implemented using conventional standard methods known to those skilled in the art, except where specifically described otherwise. These examples are illustrative and do not limit the present invention.

[0342] The immunogenic composition described in the present invention can be used in various therapeutic or prophylactic methods for the prevention, treatment, or amelioration of bacterial infections, diseases, or conditions in a subject. In particular, the immunogenic composition described in the present invention can be used to prevent, treat, or ameliorate Streptococcus pneumoniae infections, diseases, or conditions in a subject.

[0343] In one embodiment, the present invention provides a method for preventing, treating or ameliorating Streptococcus pneumoniae infection, diseases or conditions in a subject, comprising administering an immunologically effective dose of an immunogenic composition according to the present invention to the subject.

[0344] In some embodiments, such as those indicated above, the infection, diseases or conditions are selected from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, cerebrospinal meningitis, bacteremia, septicemia, pyothorax, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection and brain abscess.

[0345] In one embodiment, the present invention provides a method for inducing an immune response to Streptococcus pneumoniae in a subject, comprising administering an immunologically effective dose of an immunogenic composition according to the present invention to the subject.

[0346] In one embodiment, the immunogenic composition described in the present invention is intended for use as a vaccine. In one embodiment, such as those noted above, the immunogenic composition described in the present invention can be used to prevent Streptococcus pneumoniae infection. Thus, in one embodiment, the present invention provides a method for preventing Streptococcus pneumoniae infection in a subject, comprising administering an immunologically effective dose of the immunogenic composition according to the present invention to the subject.

[0347] In some embodiments, such as those indicated above, the infection is selected from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, cerebrospinal meningitis, bacteremia, septicemia, pyothorax, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, and brain abscess. In one embodiment, the subject requiring vaccination is a mammal, such as a human, a cat, a sheep, a pig, a horse, a cow, or a dog.

[0348] In one embodiment, the immunogenic composition described in the present invention is for use in a method for preventing, treating or ameliorating an infection, disease or condition associated with Streptococcus pneumoniae in a subject. In some embodiments, such as those indicated above, the infection, disease or condition is selected from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, cerebrospinal meningitis, bacteremia, septicemia, pyothorax, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection and brain abscess.

[0349] In one embodiment, the immunogenic composition described in the present invention is for use as a vaccine. In one embodiment, such as those indicated above, the immunogenic composition described in the present invention can be used to prevent Streptococcus pneumoniae infection in a subject. Thus, in one embodiment, the immunogenic composition described in the present invention is for use in a method for preventing Streptococcus pneumoniae infection in a subject. In some embodiments, such as those indicated above, the infection is selected from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, cerebrospinal meningitis, bacteremia, septicemia, pyothorax, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, and brain abscess.In one embodiment, the subject requiring vaccination is a mammal, such as a human, cat, sheep, pig, horse, cow, or dog.

[0350] The immunogenic composition of the present invention can be used to protect or treat a human susceptible to pneumococcal infection by administering the immunogenic composition systemically or transmucosally. In one embodiment, the immunogenic composition described in the present invention is administered intramuscularly, intraperitoneally, intradermally, or subcutaneously. In one embodiment, the immunogenic composition described in the present invention is administered by intramuscular, intraperitoneal, intradermal, or subcutaneous injection. In one embodiment, the immunogenic composition described in the present invention is administered by intramuscular or subcutaneous injection.

[0351] In the ELISA (enzyme-linked immunosorbent assay) method, antibodies from the vaccinated subject's serum are co-cultured with a polysaccharide adsorbed on a solid support. The bound antibody is detected using a secondary detection antibody conjugated to an enzyme.

[0352] This ELISA measures the level of type-specific IgG antibody to Streptococcus pneumoniae capsular polysaccharide (PS) present in human serum. When diluted human serum is added to a microtiter plate coated with type-specific capsular PS, the antibody specific for capsular PS binds to the microtiter plate. The antibody bound to the plate is detected using goat anti-human IgG antibody labeled with alkaline phosphatase and then the p-nitrophenyl phosphate substrate.

[0353] The optical density of the colored final product is proportional to the amount of capsular PS antibody present in the serum.

[0354] In one embodiment, an immunogenic composition comprising one or more Streptococcus pneumoniae serotype 2, 9N, 17F and 20 polysaccharide-protein conjugates can induce an IgG antibody that can bind Streptococcus pneumoniae serotype 15B polysaccharide at a concentration of at least 0.05, 0.1, 0.2, 0.3, 0.35, 0.4 or 0.5 μg / mL when measured in an ELISA assay in humans.

[0355] In one embodiment, an immunogenic composition comprising one or more Streptococcus pneumoniae serotype 2, 9N, 17F and 20 polysaccharide-protein conjugates can induce the formation of antibodies capable of phagocytosis of one or more Streptococcus pneumoniae serotypes selected from Streptococcus pneumoniae serotypes 2, 9N, 17F and 20 in an opsonin-initiated phagocytosis assay as described herein.

[0356] In one embodiment, an immunogenic composition comprising one or more Streptococcus pneumoniae serotype 2, 9N, 17F, and 20 polysaccharide-protein conjugates has a higher OPA titer compared to the OPA titer obtained for natural unconjugated Streptococcus pneumoniae capsular polysaccharide in a test conducted in an OPA assay.

[0357] The opsonin-induced pneumococcal phagocytosis assay (OPA), which measures the killing rate of Streptococcus pneumoniae cells by phagocytic cells in the presence of functional antibody and complement, is considered as an important alternative for evaluating the efficacy of pneumococcal vaccine.

[0358] The opsonin-induced phagocytosis assay (OPA) can be performed by culturing a mixture of Streptococcus pneumoniae cells, heat-inactivated serum from the person participating in the assay, differentiated HL-6 cells (phagocytes), and exogenous complement sources (e.g., rabbit complement) with each other. Opsonin-induced phagocytosis occurs during cultivation, and antibody- and complement-coated bacterial cells are killed during opsonin-induced phagocytosis. The number of colony-forming units (CFU) of surviving bacteria that evade opsonin-induced phagocytosis is measured by staining the assay mixture. The OPA titer is defined as the reciprocal dilution level that achieves a 50% reduction relative to the number of bacteria in a control well containing no test serum. The OPA titer is interpolated for 2 diluents containing the threshold amount providing 50% destruction.

[0359] In OPA, which studies cell killing, a titer of 1:8 or more is considered as the final titer, considering the amounts used.

[0360] In one embodiment, an immunogenic composition comprising one or more Streptococcus pneumoniae serotype 2, 9N, 17F and 20 polysaccharide-protein conjugates can induce a titer of at least 1:8 against one or more Streptococcus pneumoniae serotypes selected from 2, 9N, 17F and 20 as measured in an opsonin-induced phagocytosis (OPA) cell killing assay. In one embodiment, an immunogenic composition comprising one or more polysaccharide conjugates of Streptococcus pneumoniae serotypes 2, 9N, 17F and 20 can induce a titer of at least 1:8 against Streptococcus pneumoniae serotypes 2, 9N, 17F and 20 in at least 60%, 70%, 80%, 90% or at least 93% of subjects as measured in an opsonin-induced phagocytosis (OPA) cell killing assay.

[0361] 6. Subject treated with the immunogenic composition according to the present invention

[0362] As described in the present invention, the immunogenic composition described in the present invention can be used in various therapeutic and prophylactic methods for preventing, treating or alleviating bacterial infection, diseases or conditions in a subject.

[0363] In a preferred embodiment, the subject is a human. In a most preferred embodiment, the subject is a newborn (i.e., 3 months or younger), an infant (i.e., 3 months to 1 year old), or a toddler (i.e., 1 year to 4 years old).

[0364] In one embodiment, the immunogenic composition described in the present invention is intended for use as a vaccine. In an embodiment such as that described above, the subject requiring vaccination may be under 1 year of age. For example, the subject requiring vaccination is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 months old. In one embodiment, the subject requiring vaccination is about 2, about 4, or about 6 months old. In another embodiment, the subject requiring vaccination is under 2 years of age. For example, the subject requiring vaccination is between about 12 months and about 15 months old.In some cases, a single dose of the immunogenic composition of the present invention may be required, but in some circumstances, a second, third or fourth dose may be provided.

[0365] In one embodiment, the subject requiring vaccination is an adult over 50 years of age, more preferably an adult over 55 years of age.

[0366] In one embodiment, the subject requiring vaccination is an adult over 65 years of age, 70 years of age, 75 years of age, or 80 years of age.

[0367] In one embodiment, the subject requiring vaccination is an immunocompromised individual, in particular a human. An immunocompromised individual is generally defined as a person with a reduced or diminished ability to mount normal bodily fluid- or cell-mediated defense against infectious agents.

[0368] In one embodiment of the present invention, an immunocompromised subject requiring vaccination suffers from diseases or conditions that damage the immune system and produces an antibody response that is insufficient to protect against pneumococcal disease or to treat diseases.

[0369] In one embodiment, the diseases are primary immunodeficiency diseases. Preferably, the primary immunodeficiency diseases are selected from the group consisting of complex T- and B-cell immunodeficiency, antibody deficiency, Wells syndrome, immune dysregulation diseases, phagocyte-mediated diseases, congenital deficiencies, autoinflammatory diseases, and complement deficiency.

[0370] In a specific embodiment of the present invention, an immunocompromised subject requiring vaccination may suffer from a disease selected from the group consisting of: HIV infection, acquired immunodeficiency syndrome (AIDS), cancer, chronic heart or lung disease, congestive heart failure, diabetes, chronic liver disease, alcohol dependence, cirrhosis, cerebrospinal fluid leak, cardiomyopathy, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), spleen dysfunction (e.g., sickle cell anemia), spleen insufficiency (asplenia), hematological malignancy, leukemia, multiple myeloma, Hodgkin's lymphoma, lymphoma, renal failure, nephrotic syndrome, and asthma.

[0371] In one embodiment of the present invention, an immunocompromised subject requiring vaccination may suffer from a nutritional disorder.

[0372] In a specific embodiment of the present invention, an immunocompromised subject requiring vaccination may receive a drug or undergo treatment that reduces the body's resistance to infection.

[0373] In a specific embodiment of the present invention, the immunocompromised subject requiring vaccination may be a smoker.

[0374] In a specific embodiment of the present invention, the white blood cell count (leukocyte count) of an immunocompromised subject requiring vaccination may be 5 x 10 9 cells / liter or less or 4 x 10 9 cells / liter or less, or 3 x 10 9 cells / liter or less, or 2 x 10 9 cells / liter or less, or 1 x 10 9 cells / liter or less, or 0.5 x 10 9 cells / liter or less, or 0.3 x 10 9 cells / liter or less, or 0.1 x 10 9 cells / liter or less.

[0375] White Blood Cell Count (WBC): The number of white blood cells (WBC) in the blood. WBC is measured as part of a complete blood count (CBC). White blood cells are infection-fighting cells and are different from red blood cells (which carry oxygen).

[0376] There are different types of white blood cells, such as neutrophils (polymorphonuclear white blood cells; PMN), basal cells (some immature neutrophils), T lymphocytes (T cells), B lymphocytes (B cells), monocytes, eosinophils, and basophils. All of the above types of white blood cells are included in the total white blood cell count. The normal range of white blood cell count in most cases is between 4,300 and 10,800 cells / cubic milliliter of blood. It is also called the white blood cell count, and in the international system of measurement, it can be indicated as 4.3 - 10.8 x 10 9 / liter.

[0377] In a specific embodiment of the present invention, the immunocompromised subject requiring vaccination suffers from neutropenia. In a specific embodiment of the present invention, the neutrophil count of the immunocompromised subject requiring vaccination may be 2 x 10 9 cells / liter or less or 1 x 10 9 cells / liter or less, or 0.5 x 10 9 cells / liter or less, or 0.1 x 10 9 cells / liter or less, or 0.05 x 10 9 cells / liter or less.

[0378] Low white blood cell count, or "neutropenia," is a condition characterized by abnormally low levels of neutrophils in the bloodstream. Neutrophils are a unique type of white blood cell that help prevent infection and make it more difficult for them to become infected. The most common reason cancer patients suffer from neutropenia is the side effects of chemotherapy. Chemotherapy-induced neutropenia increases the risk of infection in patients and leads to interruptions in cancer treatment.

[0379] In a specific embodiment of the present invention, the CD4+ cell count of an immunocompromised subject requiring vaccination may be 500 / mm 3 or less or 300 / mm 3 or less, or 200 / mm 3 or less, or 100 / mm 3 or less, or 75 / mm 3 or less, or 50 / mm 3 or less.

[0380] CD4 cell count results are usually reported as the number of cells per mm 3 The normal CD4 count is between 500 and 1600, and the CD8 count is between 375 and 1100. The CD4 count is significantly reduced in people with HIV.

[0381] In one embodiment of the present invention, the immunodeficient subject described herein may be a man or a woman.

[0382] 7. Prescribed administration

[0383] In some cases, a single dose of the immunogenic composition according to the present invention may be required, but in certain conditions, such as those with more severe immunodeficiency, a second, third, or fourth dose may be administered. Following the initial vaccination, the subject may be given one or more additional immunizations at appropriate intervals.

[0384] In one embodiment, the vaccination regimen with the immunogenic composition according to the present invention comprises a single dose. In a specific embodiment, the single-dose regimen is intended for a healthy individual at least 2 years of age.

[0385] In one embodiment, the vaccination schedule with the immunogenic composition according to the present invention is a multiple-dose schedule. In a specific embodiment, the multiple-dose schedule consists of a series of 2 doses separated by an interval of from about one month to about two months. In a specific embodiment, the multiple-dose schedule consists of a series of 2 doses separated by an interval of about 1 month, or a series of 2 doses separated by an interval of about 2 months.

[0386] In another embodiment, the multiple-dose regimen consists of a series of 3 doses separated by an interval of from about 1 month to about 2 months. In another embodiment, the multiple-dose regimen consists of a series of 3 doses separated by an interval of about 1 month, or a series of 3 doses separated by an interval of about 2 months.

[0387] In another embodiment, the multiple-dose regimen consists of a series of 3 doses separated by an interval of about 1 month to about 2 months, and a subsequent fourth dose administered at an interval of about 10 months to about 13 months after the first dose. In another embodiment, the multiple-dose regimen consists of a series of 3 doses separated by an interval of about 1 month, and a subsequent fourth dose administered at an interval of about 10 months to about 13 months after the first dose, or a series of 3 doses separated by an interval of about 2 months, and a subsequent fourth dose administered at an interval of about 10 months to about 13 months after the first dose.

[0388] In one embodiment, the multiple dose regimen consists of at least 1 dose (e.g., 1, 2, or 3 doses) administered at 1 year of age, followed by at least 1 dose administered during early childhood.

[0389] In one embodiment, the multiple-dose regimen consists of a series of 2 or 3 doses separated by an interval of about 1 month to about 2 months (e.g., 28 to 56 days between doses) after an initial administration at 2 months of age, and a subsequent dose administered at a toddler age of 12 to 18 months. In one embodiment, the multiple-dose regimen consists of a series of 3 doses separated by an interval of about 1 month to about 2 months (e.g., 28 to 56 days between doses) after an initial administration at 2 months of age, and a subsequent dose administered at a toddler age of 12 to 15 months. In another embodiment, the multiple-dose regimen consists of a series of 2 doses separated by an interval of about 2 months after an initial administration at 2 months of age, and a subsequent dose administered at a toddler age of 12 to 18 months.

[0390] In one embodiment, the multiple-dose regimen consists of a series of 4 doses of vaccine administered at 2, 4, 6 months, and 12 to 15 months of age.

[0391] In one embodiment, the initial dose is provided on day 0, and one or more additional doses are provided at intervals of from about 2 to about 24 weeks, wherein preferably the interval between administrations is from 4 to 8 weeks.

[0392] In one embodiment, an initial dose is provided on day 0, with an additional dose provided approximately 3 months later.

[0393] [PREFERRED EFFECTS]

[0394] The present invention provides an immunogenic conjugate for a new serotype, such as serotype 17F, which has not previously been used to provide a protective effect.

[0395] The present invention provides a polyvalent pneumococcal vaccine that provides a broad spectrum of protection by containing a conjugate of a new serotype that has not previously been used to provide a protective effect.

[0396] In addition, it is possible to form antibodies to different pneumococcal serotypes in the absence of a specific interference phenomenon, thereby providing broad-spectrum immunity.

[0397] The present invention can provide excellent antibody titer.

[0398] The polyvalent pneumococcal vaccine according to the present invention has fewer side effects.

[0399] The vaccine according to the present invention can be inoculated into infants and young preschool children, as well as the elderly.

[0400] [IMPLEMENTATION OF THE INVENTION]

[0401] The present invention will now be described in more detail using the following examples, etc. However, the examples of the present invention can be modified to form various other forms, and the scope of the present invention should not be considered as limited to the examples described below. The examples of the present invention are given to illustrate the present invention and to more accurately understand the present invention.

[0402] Example 1. Production of a vaccine based on a conjugate of a polysaccharide isolated from serotype 2, 9N, 17F or 20,-protein

[0403] [1. Polysaccharide conjugate isolated from Streptococcus pneumoniae serotype 2-protein]

[0404] Preparation of a polysaccharide-protein conjugate from Streptococcus pneumoniae serotype 2

[0405] Obtaining a mother cell bank and preparing preparations

[0406] Streptococcus pneumoniae serotype 2 was purchased from the American Type Culture Collection (ATCC) (strain ATCC 6302). Several generations of the inoculum were grown to isolate the strain and remove animal-derived components. The inoculum tube was frozen using synthetic glycerol as a cryopreservative (< -70°C). To obtain a cell bank, all cultures were proliferated in soybean-based growth medium. Before freezing, the cells were concentrated by centrifugation, the spent medium was removed, and the cell pellet was then resuspended in new medium containing a cryopreservative (e.g., synthetic glycerol).

[0407] Fermentation

[0408] To prepare the preparation, cultures isolated from a cell bank were inoculated into a seed bottle containing soybean-based growth medium. The cells were grown at a specific temperature without agitation until the desired growth level was reached. They were then inoculated from the seed bottle into a seed fermenter containing soybean-based growth medium with controlled temperature, pH, and agitation speed. After growth ceased or the fermenter's throughput threshold was reached, fermentation was stopped. After stopping fermentation by adding an inactivator, residual cellular components were removed using a combination of continuous-flow centrifugation and filtration.

[0409] Cleaning

[0410] The purification of pneumococcal polysaccharide involved multilayer filter filtration, multiple concentration / diafiltration steps, and precipitation / elution steps.

[0411] Activation

[0412] The final concentration of the polysaccharide was adjusted to approximately 2.0 g / L in 0.01 N hydrochloric acid by successively adding a calculated amount of 0.1 N hydrochloric acid and WFI (water for injection). The polysaccharide hydrolysis reaction was carried out at 60 °C for 60 minutes. After the temperature of the reaction solution was lowered to room temperature, the pH of the reaction mixture was adjusted to approximately 6.0 by adding 0.1 M sodium monohydrogen phosphate solution. After adjusting the pH, the temperature was brought to 23 °C. Oxidation was initiated by adding sodium periodate in an amount of approximately 0.023~0.114 mg per 1 mg of sugar. The oxidation reaction was carried out at 23 °C for 18 hours.

[0413] The activated polysaccharide was concentrated and diafiltered using an ultrafiltration membrane with an MWCO of 100 kDa. Diafiltration was performed using WFI at 10 times the diafiltration volume. The purified activated polysaccharide was then stored at 2 ~ 8 °C. The purified activated polysaccharide was characterized, specifically, by (i) the polysaccharide concentration determined colorimetrically, (ii) the aldehyde concentration determined colorimetrically, (iii) the oxidation state, and (iv) the molecular weight determined by SEC-MALLS.

[0414] SEC-MALLS is used to determine the molecular weight of polysaccharides and polysaccharide-protein conjugates. SEC is used to separate polysaccharides in a moving fluid flow. A refractive index (RI) and multi-angle laser light scattering detector are used to determine molecular weight. When light interacts with a substance, it scatters, and the amount of scattered light depends on the concentration, the square of the dn / dc (refractive index increment), and the molecular weight of the material. The molecular weight is calculated based on the scattered light signal readout on the MALLS detector and the concentration signal on the RI detector.

[0415] The oxidation state (DO) of the activated polysaccharide was determined as "the number of moles of repeating sugar units ÷ the number of moles of aldehyde." The number of moles of repeating sugar units was determined using various colorimetric methods, such as the anthrone method. In addition, the number of moles of aldehyde was determined using the Parke-Johnson colorimetric method.

[0416] Preferably, the activated capsular polysaccharide of Streptococcus pneumoniae serotype 2 obtained by the said method has an oxidation state of 2 to 18 and a molecular weight of about 100 kDa to 400 kDa.

[0417] Conjugation method

[0418] The activated polysaccharide was combined with sucrose at a ratio of 2 to 8 g sucrose per gram of activated polysaccharide. The bottle containing the mixture was then lyophilized. After lyophilization, the bottle containing the lyophilized activated polysaccharide was stored at -20 to -30°C. A calculated amount of CRM197 was separately lyophilized. The lyophilized CRM197 was stored at -20 to -30°C.

[0419] The lyophilized activated polysaccharide was redissolved in anhydrous dimethyl sulfoxide (DMSO) solution. After complete dissolution of the polysaccharide, anhydrous DMSO was added to redissolve the lyophilized CRM197. The redissolved activated polysaccharide and redissolved CRM197 were combined in a reaction container (introduced ratio from 0.5 to 2:1) and then thoroughly mixed. The conjugation reaction was initiated by adding 1.0 molar equivalent of sodium cyanoborohydride (NaBH3CN) to the reaction mixture. WFI was added to the reaction mixture at a target concentration of 1% (v / v), and the reaction was carried out at 23°C for 22 to 26 hours. The conjugation reaction was terminated by adding 2.0 molar equivalents of sodium borohydride (NaBH4) and WFI at a target concentration of 5% (v / v) to the reaction mixture, thereby capping the unreacted aldehyde. The capping reaction was carried out at 23°C for 4.5 hours.

[0420] The conjugate solution was diluted with 0.9% sodium chloride solution and prepared for purification by concentration and diafiltration through a membrane with a MWCO of 100 kDa. The diluted conjugate solution was passed through a 0.8 μm filter and diafiltered using 0.9% sodium chloride at 15-40 times the diafiltration volume. After completion of diafiltration, the residual solution was filtered through a 0.2 μm filter. The conjugate solution was diluted with 0.9% sodium chloride solution to a concentration of less than approximately 0.55 mg / mL and stored at 2 to 8°C after sterile filtration.

[0421] The purified serotype 2 conjugate was characterized by (i) the polysaccharide concentration determined colorimetrically, (ii) the protein concentration determined colorimetrically (Lowry), (iii) the polysaccharide to protein ratio, (iv) the molecular weight distribution determined by size exclusion chromatography (CL-4B), (iv) the free sugar content, and (v) the molecular weight determined by SEC-MALLS.

[0422] As part of the production method, changes in the characteristics of the serotype 2 conjugate were observed by adjusting the oxidation state (DO). The results are shown in Table 1.

[0423] [Table 1]

[0424] Conjugate No. 1-1 1-2 1-3 1-4 Molecular weight of activated polysaccharide, kDa 365 313 300 231 DO 14,2 8,6 6,7 2,7 Ratio of input substances (P:S) 1:1 Conjugate yield, % 72 47 57 67 Saccharide to protein ratio 1,2 1,1 1,2 1,0 Free polysaccharide, % 27 13 8 1 Molecular weight distribution, % 93 92 88 64 Molecular weight of the conjugate, kDa 4918 3845 3485 1988

[0425] In this production method, changes in the characteristics of the serotype 2 conjugate were observed by adjusting the ratio of activated polysaccharides to CRM197 used during lyophilization. The results are presented in Table 2.

[0426] [Table 2]

[0427] Conjugate No. 1-5 1-6 1-7 1-8 1-9 1-10 1-11 1-12 1-13 1-14 Molecular weight of activated polysaccharide, kDa 260 147 DO 9,4 3,0 Ratio of input substances (P:S) 2:1 1,5:1 1:1 0,7:1 0,5:1 2:1 1,5:1 1:1 0,7:1 0,5:1 Conjugate yield, % 47 53 57 58 58 64 70 70 73 68 Saccharide to protein ratio 0,53 0,69 0,99 1,44 1,81 0,58 0,69 1,04 1,41 1,86 Free polysaccharide, % 14 15 18 26 27 1 0 1 9 14 Molecular weight distribution, % 94 96 97 96 94 76 71 67 65 63 Molecular weight of the conjugate, kDa 15819 11389 5532 3544 2629 8255 4070 2185 1226 1050

[0428] Immunogenicity study of serotype 2 polysaccharide-protein conjugate

[0429] A monovalent conjugate composition was obtained containing a conjugate of Streptococcus pneumoniae serotype 2 polysaccharide conjugated with CRM197 protein.

[0430] The immunogenicity of the monovalent immunogenic compositions shown in Table 1 and Table 2 was analyzed using ELISA in rabbits, and thereby the serotype-specific IgG concentration in serum was measured.

[0431] A group of five female New Zealand White rabbits weighing 2.5–3.5 kg were immunized intramuscularly at week 0 with the intended human clinical dose (conjugate, 2.2 μg; + aluminum, 0.25 mg / ml as AlPO4). The rabbits were additionally immunized at week 2 with the same dose of the conjugate vaccine, and blood was then collected at week 4. Serum samples collected at weeks 0 and 4 were analyzed for serotype-specific ELISA.

[0432] The results of the analysis are shown in Table 3. Rabbits immunized with the monovalent conjugate composition (conjugate No. 1-6) showed a significant increase in total IgG titer against serotype 2. Rabbits immunized with the other conjugate showed a significant increase in total IgG titer.

[0433] The values ​​shown in the following Table 3 correspond to the measured values ​​of IgG concentration after immunization with conjugate No. 1-6 shown in Table 1.

[0434] [Table 3]

[0435] IgG concentration (IU / ml) Serotype Before immunization After immunization 2 130,0 62164,7

[0436] [2. Polysaccharide conjugate isolated from serotype 9N Streptococcus pneumoniae-protein]

[0437] Preparation of a polysaccharide conjugate isolated from serotype 9N of Streptococcus pneumoniae - protein

[0438] Obtaining a mother cell bank and preparing preparations

[0439] Streptococcus pneumoniae serotype 9N was obtained from the American Type Culture Collection (ATCC) (strain ATCC 6309). It was processed in the same way as serotype 2.

[0440] Fermentation

[0441] Conducted in the same way as in the case of serotype 2.

[0442] Cleaning

[0443] Conducted in the same way as in the case of serotype 2.

[0444] Activation

[0445] A final polysaccharide concentration of approximately 2.0 g / L was achieved by adding the calculated amount of WFI. The pH of the reaction mixture was adjusted to approximately 6.0 if necessary. After adjusting the pH, the reaction temperature was raised to 23°C. Oxidation was initiated by adding sodium periodate at a rate of 0.024~0.189 mg per approximately 1 mg of sugar. The oxidation reaction was carried out at 23°C for 18 hours.

[0446] The activated polysaccharide was concentrated and diafiltered using an ultrafiltration membrane with an MWCO of 100 kDa. Diafiltration was performed using WFI at 10 times the diafiltration volume. The purified activated polysaccharide was then stored at 2 ~ 8 °C. The purified activated polysaccharide was characterized, specifically, by (i) the polysaccharide concentration determined colorimetrically, (ii) the aldehyde concentration determined colorimetrically, (iii) the oxidation state, and (iv) the molecular weight determined by SEC-MALLS.

[0447] SEC-MALLS is used to determine the molecular weight of polysaccharides and polysaccharide-protein conjugates. SEC is used to separate polysaccharides in a moving fluid flow. A refractive index (RI) and multi-angle laser light scattering detector are used to determine molecular weight. When light interacts with a substance, it scatters, and the amount of scattered light depends on the concentration, the square of the dn / dc (refractive index increment), and the molecular weight of the material. The molecular weight is calculated based on the scattered light signal readout on the MALLS detector and the concentration signal on the RI detector.

[0448] The oxidation state (DO) of the activated polysaccharide was determined as "the number of moles of repeating sugar units ÷ the number of moles of aldehyde." The number of moles of repeating sugar units was determined using various colorimetric methods, such as the anthrone method. In addition, the number of moles of aldehyde was determined using the Parke-Johnson colorimetric method.

[0449] The preferably activated capsular polysaccharide of Streptococcus pneumoniae serotype 9N obtained by the said method had an oxidation state of 2 to 19 and a molecular weight of about 200 kDa to 700 kDa.

[0450] Conjugation method

[0451] The activated polysaccharide was combined with the carrier protein, CRM197, at a ratio of 0.5 to 2 grams of CRM197 per gram of activated polysaccharide. The mixture was then lyophilized. After lyophilization, the lyophilized mixture of activated polysaccharide and CRM197 was stored at -20°C.

[0452] The lyophilized mixture of activated polysaccharide and CRM197 was redissolved in a 0.1 M sodium phosphate solution and then properly stirred. The final concentration of the polysaccharide in the reaction solution was approximately 10 to 20 g / L. Conjugation was initiated by adding 1.2 molar equivalents of sodium cyanoborohydride (NaBH3CN) to the mixture and reacting at 37°C for 48 hours. The conjugation reaction was terminated by adding a 0.9% sodium chloride solution in the same volume as the reaction solution in which the conjugation was carried out, and then 2.0 molar equivalents of sodium borohydride (NaBH4) were added to cap the unreacted aldehyde. The capping reaction was carried out at 23°C for 4.5 hours.

[0453] The conjugate solution was diluted with 0.9% sodium chloride solution and prepared for purification by concentration and diafiltration on a membrane with an MWCO of 100 kDa. The diluted conjugate solution was passed through a 0.45 μm filter and purified by concentration and diafiltration. Diafiltration was performed on a membrane with an MWCO of 100 kDa using 0.9% sodium chloride solution at 15-40 times the diafiltration volume. After completion of diafiltration, the residual solution was filtered through a 0.2 μm filter. The conjugate solution was diluted to a concentration of less than approximately 0.55 mg / mL and stored at 2 to 8°C after sterile filtration.

[0454] The purified serotype 9N conjugate was specifically characterized by (i) the polysaccharide concentration determined colorimetrically, (ii) the protein concentration determined colorimetrically (by Lowry), (iii) the polysaccharide to protein ratio, (iv) the molecular weight distribution determined by size exclusion chromatography (CL-4B), (iv) the free sugar content, and (v) the molecular weight determined by SEC-MALLS.

[0455] As part of the production method, changes in the characteristics of the serotype 9N conjugate were observed by adjusting the oxidation state (DO). The results are presented in Table 4.

[0456] [Table 4]

[0457] Conjugate No. 2-1 2-2 2-3 2-4 2-5 2-6 Molecular weight of activated polysaccharide, kDa 582 619 459 563 490 427 DO 18,2 9,4 7,4 6,7 4,3 2,3 Input:S ratio 0,8:1 Concentration of polysaccharide in the reaction solution in which conjugation was carried out, g / l 20,0 Conjugate yield, % 53 43 39 32 33 39 Saccharide to protein ratio 2,1 1,5 1,3 1,1 1,0 0,78 Free polysaccharide, % 44 28 22 20 21 31 Molecular weight distribution, % 52 49 50 55 44 31 Molecular weight of the conjugate, kDa 860 1110 1912 1168 1189 1160

[0458] During the production process, changes in the characteristics of the serotype 9N conjugate were observed by adjusting the mixing ratio of activated CRM197 polysaccharides during lyophilization. The results are presented in Table 5.

[0459] [Table 5]

[0460] Conjugate No. 2-7 2-8 2-9 2-10 2-11 Molecular weight of activated polysaccharide, kDa 287 DO 5,6 Input:S Ratio (P:S) 2:1 1,5:1 1:1 0,67:1 0,5:1 Concentration of polysaccharide in the reaction solution in which conjugation was carried out, g / l 20,0 Conjugate yield, % 25 50 43 41 66 Saccharide to protein ratio 0,71 0,85 1,0 1,2 1,8 Free polysaccharide, % 5 6 15 27 62 Molecular weight distribution, % 52 58 50 40 22 Molecular weight of the conjugate, kDa 3720 3713 1327 1016 545

[0461] During the production process, changes in the characteristics of the serotype 9N conjugate were observed by adjusting the polysaccharide concentration in the reaction solution used for conjugation. The results are presented in Table 6.

[0462] [Table 6]

[0463] Conjugate No. 2-12 2-13 2-14 2-15 2-16 Molecular weight of activated polysaccharide, kDa 560 DO 6,1 Input:S ratio 0,8:1 Concentration of polysaccharide in the reaction solution in which conjugation was carried out, g / l 10,0 12,5 15,0 17,5 20,0 Conjugate yield, % 20 31 28 40 42 Saccharide to protein ratio 1,0 1,0 0,93 0,99 0,97 Free polysaccharide, % 32 30 22 21 18 Molecular weight distribution, % 17 27 40 47 54 Molecular weight of the conjugate, kDa 560 546 845 932 1438

[0464] Study of the immunogenicity of the serotype 9N polysaccharide-protein conjugate

[0465] A monovalent conjugate composition containing a conjugate of a polysaccharide of serotype 9N Streptococcus pneumoniae conjugated with a CRM197 protein was obtained.

[0466] The immunogenicity of the monovalent immunogenic compositions shown in Tables 4-6 was analyzed using ELISA in rabbits, and thereby the serotype-specific IgG concentration in serum was measured.

[0467] A group of female New Zealand White rabbits were immunized intramuscularly in the same manner as for serotype 2.

[0468] The results of the analysis are shown in Table 7. Rabbits immunized with the monovalent conjugate composition (conjugate No. 2-8) showed a significant increase in total IgG titer against serotype 9N. Rabbits immunized with the other conjugate showed a significant increase in total IgG titer.

[0469] The values ​​shown in the following Table 7 correspond to the measured values ​​of IgG concentration after immunization with conjugate No. 2-8 shown in Table 5.

[0470] [Table 7]

[0471] IgG concentration (IU / ml) Serotype Before immunization After immunization 9N 130,0 656345,3

[0472] [3. Polysaccharide conjugate isolated from Streptococcus pneumoniae serotype 17F-protein]

[0473] Preparation of a polysaccharide conjugate isolated from Streptococcus pneumoniae serotype 17F-protein

[0474] Obtaining a mother cell bank and preparing preparations

[0475] Streptococcus pneumoniae serotype 17F was obtained from the American Type Culture Collection (ATCC) (strain ATCC 6317). To isolate the strain and remove animal-derived components, several generations of seed were grown. This was done in the same manner as for serotype 2.

[0476] Fermentation

[0477] Conducted in the same way as in the case of serotype 2.

[0478] Cleaning

[0479] Conducted in the same way as in the case of serotype 2.

[0480] Activation

[0481] The final concentration of the polysaccharide was adjusted to approximately 2.0 g / L in 0.01 N hydrochloric acid by successively adding calculated amounts of 0.1 N hydrochloric acid and WFI. The polysaccharide was hydrolyzed at approximately 60 °C for 60 minutes. After the temperature of the reaction solution was lowered to room temperature, the pH of the reaction mixture was adjusted to approximately 6.0 by adding 0.1 M sodium monohydrogen phosphate. After adjusting the pH, the temperature was raised to 23 °C. Oxidation was initiated by adding sodium periodate in an amount of approximately 0.008~0.219 mg per 1 mg of sugar. The oxidation reaction was carried out at 23 °C for 18 hours.

[0482] The activated polysaccharide was concentrated and diafiltered using an ultrafiltration membrane with an MWCO of 100 kDa. Diafiltration was carried out using WFI at 10 times the diafiltration volume. The purified activated polysaccharide was then stored at 2 ~ 8 °C. The purified activated polysaccharide was characterized, specifically, by (i) the polysaccharide concentration determined colorimetrically, (ii) the aldehyde concentration determined colorimetrically, (iii) the oxidation state, and (iv) the molecular weight determined by SEC-MALLS.

[0483] SEC-MALLS is used to determine the molecular weight of polysaccharides and polysaccharide-protein conjugates. SEC is used to separate polysaccharides in a moving fluid flow. A refractive index (RI) and multi-angle laser light scattering detector are used to determine molecular weight. When light interacts with a substance, it scatters, and the amount of scattered light depends on the concentration, the square of the dn / dc (refractive index increment), and the molecular weight of the material. The molecular weight is calculated based on the scattered light signal readout on the MALLS detector and the concentration signal on the RI detector.

[0484] The oxidation state (DO) of the activated polysaccharide was determined as "the number of moles of repeating sugar units ÷ the number of moles of aldehyde." The number of moles of repeating sugar units was determined using various colorimetric methods, such as the anthrone method. In addition, the number of moles of aldehyde was determined using the Parke-Johnson colorimetric method.

[0485] The preferably activated capsular polysaccharide of Streptococcus pneumoniae serotype 17F obtained by the said method had an oxidation state of 1 to 22 and a molecular weight of about 400 kDa to 900 kDa.

[0486] Conjugation method

[0487] The activated polysaccharide was combined with the carrier protein, CRM197, at a ratio of 1.0 gram of CRM197 per gram of activated polysaccharide. The mixture was then lyophilized. After lyophilization, the lyophilized mixture of activated polysaccharide and CRM197 was stored at -20°C.

[0488] The lyophilized mixture of activated polysaccharide and CRM197 was redissolved in 0.1 M sodium phosphate solution (pH 7.2 ± 0.1). The final concentration of polysaccharide in the reaction solution ranged from 15.0 to 25.0 g / L. Conjugation was initiated by adding 1.2 molar equivalents of sodium cyanoborohydride (NaBH3CN) to the mixture, and the reaction was carried out at 37°C for 48 hours. The conjugation reaction was terminated by adding 0.9% sodium chloride solution in the same volume as the reaction solution in which the conjugation was carried out, and then 2.0 molar equivalents of sodium borohydride (NaBH4) were added to block the unreacted aldehyde. The blocking reaction was carried out at 23°C for 4.5 hours.

[0489] The conjugate solution was diluted with 0.9% sodium chloride solution, preparing it for purification by concentration and diafiltration on a membrane with an MWCO of 100 kDa. The diluted conjugate solution was passed through a 0.45 μm filter and purified by concentration and diafiltration. Diafiltration was performed on a membrane with an MWCO of 100 kDa using 0.9% sodium chloride solution at 15-40 times the diafiltration volume. After completion of the primary diafiltration, the residual solution was filtered through a 0.2 μm filter and stored at 2 to 8°C.

[0490] The purified serotype 17F conjugate was specifically characterized by (i) the polysaccharide concentration determined colorimetrically, (ii) the protein concentration determined colorimetrically (by Lowry), (iii) the polysaccharide to protein ratio, (iv) the molecular weight distribution determined by size exclusion chromatography (CL-4B), (iv) the free sugar content, and (v) the molecular weight determined by SEC-MALLS.

[0491] During the production process, changes in the characteristics of the serotype 17F conjugate were observed by adjusting the oxidation state (DO) and the concentration of the polysaccharide in the reaction solution. The results are presented in Table 8.

[0492] [Table 8]

[0493] Conjugate No. 3-1 3-2 3-3 3-4 3-5 3-6 3-7 3-8 3-9 Molecular weight of activated polysaccharide, kDa 551 560 577 628 801 DO 21,3 9,4 7,5 3,8 1,3 Input:S ratio 1:1 Concentration of polysaccharide in the reaction solution in which conjugation was carried out, g / l 20,0 15,0 17,5 20,0 22,5 25,0 Conjugate yield, % 31 48 58 35 28 25 28 37 37 Saccharide to protein ratio 14,9 5,3 4,9 2,8 0,68 0,65 0.67 0.70 0.71 Free polysaccharide, % 84 82 78 60 8 4 4 6 4 Molecular weight distribution, % - 18 38 48 49 58 Molecular weight of the conjugate, kDa 372 706 456 1064 1346 2115 2531 3150 4423

[0494] Study of the immunogenicity of the serotype 17F polysaccharide-protein conjugate

[0495] A monovalent conjugate composition containing a conjugate of Streptococcus pneumoniae serotype 17F polysaccharide-protein conjugated to CRM197 protein was obtained.

[0496] The immunogenicity of the monovalent immunogenic composition shown in Table 8 was analyzed using ELISA in rabbits, and the serotype-specific IgG concentration in serum was thereby measured.

[0497] A group of female New Zealand White rabbits were immunized intramuscularly in the same manner as for serotype 2.

[0498] The results of the analysis are shown in Table 9. Rabbits immunized with the monovalent conjugate composition (conjugate No. 3-8) showed a significant increase in total IgG titer against serotype 17F. Rabbits immunized with the other conjugate showed a significant increase in total IgG titer.

[0499] The values ​​shown in the following Table 9 correspond to the measured values ​​of IgG concentration after immunization with conjugate No. 3-8 shown in Table 8.

[0500] [Table 9]

[0501] IgG concentration (IU / ml) Serotype Before immunization After immunization 17F 130,0 227590,3

[0502] [4. Streptococcus pneumoniae serotype 20 polysaccharide-protein conjugate]

[0503] Preparation of a polysaccharide conjugate isolated from Streptococcus pneumoniae serotype 20-protein

[0504] Obtaining a mother cell bank and preparing preparations

[0505] Streptococcus pneumoniae serotype 20 was obtained from the American Type Culture Collection (ATCC) (strain ATCC 6320). Processing was carried out in the same way as for serotype 2.

[0506] Fermentation

[0507] Conducted in the same way as in the case of serotype 2.

[0508] Cleaning

[0509] Conducted in the same way as in the case of serotype 2.

[0510] Activation

[0511] The final polysaccharide concentration was adjusted to approximately 2.0 g / L in 0.01 N hydrochloric acid by successively adding the calculated amount of 0.1 N hydrochloric acid and WFI. Oxidation was initiated by adding sodium periodate at a rate of approximately 0.010~0.038 mg per 1 mg of sugar. The oxidation reaction was carried out at 23°C for 18 hours.

[0512] The activated polysaccharide was concentrated and diafiltered using an ultrafiltration membrane with an MWCO of 100 kDa. Diafiltration was carried out using WFI at 10 times the diafiltration volume. The purified activated polysaccharide was then stored at 2 ~ 8 °C. The purified activated polysaccharide was characterized, specifically, by (i) the polysaccharide concentration determined colorimetrically, (ii) the aldehyde concentration determined colorimetrically, (iii) the oxidation state, and (iv) the molecular weight determined by SEC-MALLS.

[0513] SEC-MALLS is used to determine the molecular weight of polysaccharides and polysaccharide-protein conjugates. SEC is used to separate polysaccharides in a moving fluid flow. A refractive index (RI) and multi-angle laser light scattering detector are used to determine molecular weight. When light interacts with a substance, it scatters, and the amount of scattered light depends on the concentration, the square of the dn / dc (refractive index increment), and the molecular weight of the material. The molecular weight is calculated based on the scattered light signal readout on the MALLS detector and the concentration signal on the RI detector.

[0514] The oxidation state (DO) of the activated polysaccharide was determined as "the number of moles of repeating sugar units ÷ the number of moles of aldehyde." The number of moles of repeating sugar units was determined using various colorimetric methods, such as the anthrone method. In addition, the number of moles of aldehyde was determined using the Parke-Johnson colorimetric method.

[0515] Preferably, the activated capsular polysaccharide of Streptococcus pneumoniae serotype 20 obtained by the said method had an oxidation state of 4 to 16 and a molecular weight of about 400 kDa to 800 kDa.

[0516] Conjugation method

[0517] The activated polysaccharide was combined with the carrier protein, CRM197, at a ratio of 1.0 gram of CRM197 per gram of activated polysaccharide. The mixture was then lyophilized. After lyophilization, the lyophilized mixture of activated polysaccharide and CRM197 was stored at -20°C.

[0518] The lyophilized mixture of activated polysaccharide and CRM197 was redissolved in 0.1 M sodium phosphate solution (pH 7.2 ± 0.1). The final concentration of polysaccharide in the reaction solution was 15.0 g / L. Conjugation was initiated by adding 1.2 molar equivalents of sodium cyanoborohydride (NaBH3CN) to the mixture, and the reaction was carried out at 37°C for 48 hours. The conjugation reaction was terminated by adding 0.9% sodium chloride solution in the same volume as the reaction solution in which the conjugation was carried out, and then 2.0 molar equivalents of sodium borohydride (NaBH4) were added, thereby capping the unreacted aldehyde. The capping reaction was carried out at 23°C for 4.5 hours.

[0519] The conjugate solution was diluted with 0.9% sodium chloride solution and prepared for purification by concentration and diafiltration on a membrane with an MWCO of 100 kDa. The diluted conjugate solution was passed through a 0.45 μm filter and a two-step purification was performed by concentration and diafiltration. Diafiltration was carried out on a membrane with an MWCO of 100 kDa using a 0.9% sodium chloride solution at 20 times the diafiltration volume. After completion of the primary diafiltration, the residual solution was filtered through a 0.2 μm filter and stored at 2 to 8°C. The conjugate solution was diluted to a concentration of less than approximately 0.55 mg / mL and stored at 2 to 8°C after sterile filtration.

[0520] The purified serotype 20 conjugate was specifically characterized by (i) the polysaccharide concentration determined colorimetrically, (ii) the protein concentration determined colorimetrically (by Lowry), (iii) the polysaccharide to protein ratio, (iv) the molecular weight distribution determined by size exclusion chromatography (CL-4B), (iv) the free sugar content, and (v) the molecular weight determined by SEC-MALLS.

[0521] As part of the production method, changes in the characteristics of the serotype 20 conjugate were observed by adjusting the oxidation state (DO). The results are presented in Table 10.

[0522] [Table 10]

[0523] Conjugate No. 4-1 4-2 4-3 4-4 4-5 4-6 4-7 Molecular weight of activated polysaccharide, kDa 651 749 675 463 613 444 712 DO 15,7 15,4 8,9 7,3 6,7 4,8 4,6 Input:S ratio 1:1 Conjugate yield, % 65 57 56 49 45 24 20 Saccharide to protein ratio 3,7 2,9 2,5 2,7 2,2 2,1 1,5 Free polysaccharide, % 29 28 16 15 16 11 8 Molecular weight distribution, % 84 - 79 78 82 78 - Molecular weight of the conjugate, kDa 1968 1271 3349 2458 3645 2123 2563

[0524] Study of the immunogenicity of Streptococcus pneumoniae serotype 20 polysaccharide-protein conjugate

[0525] A monovalent conjugate composition containing a conjugate of Streptococcus pneumoniae serotype 20 polysaccharide conjugated with CRM197 was obtained.

[0526] The immunogenicity of the monovalent immunogenic composition shown in Table 10 was analyzed using ELISA in rabbits, and the serotype-specific IgG concentration in serum was thereby measured.

[0527] Female New Zealand White rabbits were immunized intramuscularly in the same manner as for serotype 2.

[0528] The results of the analysis are shown in Table 11. Rabbits immunized with the monovalent conjugate composition (conjugate No. 4-7) showed a significant increase in total IgG titer against serotype 20. Rabbits immunized with the other conjugate showed a significant increase in total IgG titer.

[0529] The values ​​shown in the following Table 11 correspond to the measured values ​​of IgG concentration after immunization with conjugate No. 4-7 shown in Table 10.

[0530] [Table 11]

[0531] IgG concentration (IU / ml) Serotype Before immunization After immunization 20 166,2 277210,1

[0532] Example 2. Preparation of a polyvalent Streptococcus pneumoniae polysaccharide-protein conjugate

[0533] [5. 15-valent Streptococcus pneumoniae polysaccharide-protein conjugate]

[0534] Preparation of a 15-valent Streptococcus pneumoniae polysaccharide-protein conjugate

[0535] A 15-valent conjugate composition was prepared containing a polysaccharide-protein conjugate isolated from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F and 23F, in all cases the polysaccharides were conjugated with CRM197 (15vPnC).

[0536] For serotypes 2 and 9N, the conjugate was obtained in the above-mentioned manner, and for other serotypes, the conjugate was obtained according to the method described in Korean Patent Application No. 2012-0065893.

[0537] The required volume of final bulk concentrate was calculated based on the batch volume and the saccharide concentration of the bulk concentrate. The required amounts of 0.85% sodium chloride, polysorbate 80, and succinate buffer were added to a pre-labeled mixing container, followed by the bulk concentrate. The mixture was mixed well and filtered through a 0.22 μm filter. The resulting bulk solution was slowly stirred during and after the addition of bulk aluminum phosphate. The pH was checked and adjusted as needed. The resulting bulk product was stored at 2 to 8°C. The resulting vaccine composition contained 2.2 μg of each saccharide, but 4.4 μg of 6B; approximately 32 μg of CRM 197 carrier protein; 0.125 mg aluminum as an adjuvant (0.5 mg aluminum phosphate); approximately 4.25 mg sodium chloride; approximately 295 mcg succinate buffer; and approximately 100 mcg polysorbate 80 in a total volume of 0.5 mL.

[0538] Immunogenicity study of 15-valent Streptococcus pneumoniae polysaccharide-protein conjugate

[0539] IgG concentration measurement

[0540] The immunogenicity of the 15-valent immunogenic composition was analyzed in rabbits using ELISA, and the serotype-specific IgG concentration in serum was measured.

[0541] A group of six female New Zealand White rabbits weighing 2.5 to 3.5 kg were immunized intramuscularly at week 0 with the intended human clinical dose (2.2 μg conjugate, except for serotype 6b, for which 4.4 μg was determined; + 0.25 mg / mL aluminum as AlPO4). The rabbits were further immunized at week 3 with the same dose of conjugate vaccine, and blood samples were then collected at 3-week intervals. Serotype-specific ELISA was performed on serum samples at each week.

[0542] The serotype-specific immune response to the vaccine formulation of the present invention and the vaccine formulation of the comparative example was evaluated by IgG ELISA. The results of the analysis are shown in Table 12. The IgG concentration values ​​(IU / ml) at different time points after inoculation are shown. It was shown that rabbits immunized with 15vPnC produced antibodies to serotypes 2 and 9N, which could not be produced by Prevenar 13, and, in particular, 15vPnC could induce an equivalent or higher IgG titer in serum compared with Prevenar 13, even though its valence increased as a result of the addition of the serotype.

[0543] [Table 12]

[0544] ELISA Prevenar 13 SK-15 Type Day 0 Day 21 Day 42 Day 63 Day 0 Day 21 Day 42 Day 63 1 0 8522 14187 10207 0 2053 17110 7752 2 - - - - 134 47864 36482 23790 3 0 968 7229 5330 0 2084 11069 10947 4 0 3831 23100 16654 0 2592 15000 10179 5 97 5597 13083 14819 111 4866 19615 16062 6A 0 15810 28609 20581 0 3634 20313 10141 6B 0 12920 43575 34932 0 4296 29763 14204 7F 0 48129 26694 18014 0 43979 21590 10878 9N - - - - 0 9197 17085 13021 9V 436 17281 35392 9442 373 15128 12143 12304 14 194 8365 10403 10786 416 7066 13160 16043 18C 0 19101 23989 24862 0 19385 14192 14565 19A 0 71160 136042 139273 0 24713 64138 59135 19F 0 40695 53165 56443 0 8382 38304 30852 23F 0 11171 62331 47522 0 7116 65352 67085

[0545] OPA Analysis Results

[0546] To confirm the possible induction of a functional antibody response using the 15-valent polysaccharide-protein conjugate, a multiplex opsonophagocytic killing assay (MOPA) was performed.

[0547] Since equal amounts of serum were collected from each subject, serum samples were pooled into groups of equal size. Streptococcus pneumoniae was grown in THY medium for each serum sample and diluted to 1000 CFU / 10 μl. 200 μl of opsonization buffer, 10 μl of diluted serum, and 10 μl of diluted Streptococcus pneumoniae were mixed and reacted at room temperature for 1 hour. A mixed solution of predifferentiated HL-60 cells and complement was added, and the reaction was carried out in a CO2 incubator (37°C) for 1 hour. Phagocytosis was interrupted by lowering the temperature, and 5 μl of the reaction solution was stained in agar medium pre-dried for 30 to 60 minutes. The material was cultured in a CO2 incubator (37°C) for 12 to 18 hours, and the number of colonies was counted. The OPA titer corresponded to the dilution level at which 50% of the cells died.As a comparison example, the 13-valent vaccine (Prevenar 13, Pfizer) was used for evaluation under the same conditions, the results are shown in Table 13.

[0548] [Table 13]

[0549] OPA Prevenar 13 SK-15 Type Day 21 Day 42 Day 63 Day 21 Day 42 Day 63 1 16 64 64 4 64 64 2 - - - 128 512 512 3 1 2 4 1 4 4 4 128 1024 1024 128 1024 1024 5 64 256 512 32 256 512 6A 512 2048 2048 256 2048 2048 6B 256 2048 2048 128 2048 2048 7F 1024 2048 2048 1024 2048 2048 9N - - - 512 2048 2048 9V 256 512 512 256 512 512 14 256 1024 1024 256 1024 1024 18C 1024 1024 2048 1024 512 2048 19A 512 1024 2048 256 1024 1024 19F 256 1024 1024 128 512 512 23F 256 2048 2048 256 2048 2048

[0550] [6. 23-valent Streptococcus pneumoniae polysaccharide-protein conjugate]

[0551] Preparation of a 23-valent Streptococcus pneumoniae polysaccharide-protein conjugate

[0552] A 23-valent conjugate composition was prepared comprising polysaccharide-protein conjugates wherein polysaccharides isolated from Streptococcus pneumoniae serotypes 1, 2, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F were conjugated with CRM197, and polysaccharide-protein conjugates wherein Streptococcus pneumoniae serotypes 3 and 5 were conjugated with TT (tetanus toxoid) (23vPnC).

[0553] In the case of serotypes 2, 9N, 17F, and 20, the conjugate was prepared in the above-mentioned manner, and in the case of other serotypes, conjugates with either CRM197 or TT were prepared according to the methods described in Korean Patent Application No. 2012-0065893, U.S. Patent Application Nos. 62 / 371529, 62 / 371553, and 62 / 626482.

[0554] The required volume of final bulk concentrate was calculated based on the batch volume and the saccharide concentration of the bulk concentrate. The required amounts of 0.85% sodium chloride, polysorbate 80, and succinate buffer were added to a pre-labeled mixing container, followed by the bulk concentrate. The mixture was mixed thoroughly and filtered through a 0.22 μm filter. The resulting bulk solution was slowly stirred during and after the addition of bulk aluminum phosphate. The pH was checked and adjusted as needed. The resulting bulk product was stored at 2 to 8°C. The resulting vaccine composition contained 2.2 μg of each saccharide, but 4.4 μg of 6B; approximately 50 to 85 μg of carrier protein; experimental amount of aluminum as an adjuvant (e.g. in the case of group 4 given in Table 14, 0.125 mg of elemental aluminum, i.e.0.5 mg aluminum phosphate); approximately 4.25 mg sodium chloride; approximately 295 mcg succinate buffer; and approximately 100 mcg polysorbate 80 for a total volume of 0.5 mL.

[0555] Immunogenicity study of 23-valent Streptococcus pneumoniae polysaccharide-protein conjugate

[0556] IgG concentration measurement

[0557] The immunogenicity of a 23-valent immunogenic composition was analyzed in rabbits using ELISA, thereby measuring serotype-specific IgG concentrations in serum. The ability of the 23vPnC vaccine containing an adjuvant to induce a serotype-specific immune response was studied.

[0558] A group of 5 female New Zealand White rabbits weighing 2.5 kg to 3.5 kg were immunized intramuscularly at week 0 with the intended human clinical dose (2.2 μg conjugate, except for serotype 6b, for which 4.4 μg was determined), in which the aluminum content as AlPO4 was 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL, respectively. The rabbits were further immunized at week 2 with the same dose of conjugate vaccine, and then blood samples were collected at week 4. Serotype-specific ELISA was performed on serum samples collected at weeks 0 and 4.

[0559] The 13-valent vaccine (Prevenar 13, Pfizer) was used as a comparison example for evaluation under the same conditions, and the analysis results are shown in Table 14. The IgG concentration values ​​(IU / ml) at 4 weeks after inoculation are shown.

[0560] The geometric mean titer (GMT) measured in a pooled set of serum samples after administration of 23vPnV and Prevenar 13 was obtained. The data obtained demonstrate that IgG antibodies are induced at an increased level when using the adjuvanted 23vPnV formulation compared to the same vaccine without adjuvant. In particular, the possibility of producing a 23-valent immunogenic vaccine containing all of serotypes 2, 9N, 17F and 20 was confirmed. As can be seen in the results presented below, in particular, antibodies to serotype 2, etc., which were not produced when using Prevenar 13, could be produced. In addition, it was confirmed that the vaccine could induce an immune response against all contained serotypes without significantly affecting the production of antibodies to antigens of other serotypes, despite the significantly increased valency.

[0561] [Table 14]

[0562] Group 1: PCV24 I aluminum 0 Control group Group 2: PCV24 I aluminum 62.5 Group 3: PCV24 I aluminum 125 Group 4: PCV24 I aluminum 250 Group 5: PCV24 I aluminum 500 Group 6: PCV24 I aluminum 1000 Group 7 (Prevenar® 13) Comparison group Type 1 4421 10951 11205 9595 12821,5 12038,5 9131,0 Type 2 4850,7 7347 7358,2 6833,1 9909,4 7875,3 - Type 3 15552,6 17362,7 23574,8 11718 24425,5 18313,2 4265,9 Type 5 5676,1 11391,8 10274,5 10699,7 13622,7 15469 5930,1 Type 6A 15265,3 13034,2 30631,9 17203,6 21335,1 21011,1 5697,2 Type 6B 3566,3 8567,3 17964,5 8101,7 21985,6 19993,7 4136,6 Type 7F 10474,8 32904,5 36935,5 28650,3 45878,8 48559 31991,5 Type 8 18684,5 26486,5 39449,2 26369,3 43372,9 56812,6 - Type 9N 29350,5 52915,1 61918,7 25465,9 73406,3 95465,2 - Type 9V 9041 19108 24479,9 24602,9 30087,5 36732,8 23053,6 Type 10A 19833,3 38644,7 39959,5 73948,6 40524,8 60464,4 - Type 11A 893,6 3845,9 4447,6 2474,3 5683,2 8358,5 - Type 12F 4785,2 10497,8 7381,7 8444,8 8113,6 14211,8 - Type 14 9177,7 17574 12811,4 15834,6 13543,4 23876,5 11178,8 Type 15B 4908,7 17293,7 18345,2 4936,3 19436,1 28797,9 - Type 17F 7441,1 9373,4 14848,8 10186,3 14319,7 32906,8 - Type 18C 16747,2 27864,6 44605,6 29416,3 37658 38860,5 34163,2 Type 19A 781,9 4088,9 5846,5 4114,6 8824,5 12216,5 14381,8 Type 19F 4501,9 32543,6 27502,3 28629,2 36290,5 68218,5 15315,5 Type 20 18252,8 32553,5 34663,5 21760,3 37978,2 44630 - Type 22F 6790,8 24687,1 24800,1 19953 42634,5 55543,4 - Type 23F 823,6 3859,8 7938 6634 9468,7 10348,8 9440,5 Type 33F 7261,6 23864,8 24843,6 20105,5 22586,9 28958 -

[0563] [7. 24-valent Streptococcus pneumoniae polysaccharide-protein conjugate]

[0564] Preparation of a 24-valent Streptococcus pneumoniae polysaccharide-protein conjugate

[0565] A 24-valent conjugate composition was prepared containing polysaccharide-protein conjugates wherein polysaccharides isolated from Streptococcus pneumoniae serotypes 2, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F were conjugated with CRM197, and polysaccharide-protein conjugates wherein Streptococcus pneumoniae serotypes 1 and 5 were conjugated with TT (tetanus toxoid) (24vPnC). For serotypes 2, 9N, 17F and 20, the conjugate was prepared as described above, and for other serotypes, the conjugate with CRM197 or TT was prepared according to the methods described in Korean Patent Application No. 2012-0065893, US Patent Applications Nos. 62 / 371529, 62 / 371553 and 62 / 626482.

[0566] The required volume of final bulk concentrate was calculated based on the batch volume and the saccharide concentration of the bulk concentrate. The required amounts of 0.85% sodium chloride, polysorbate 80, and succinate buffer were added to a pre-labeled mixing container, followed by the bulk concentrate. The mixture was mixed well and filtered through a 0.22 μm filter. The resulting bulk solution was slowly stirred during and after the addition of bulk aluminum phosphate. The pH was checked and adjusted as needed. The resulting bulk product was stored at 2 to 8°C. The resulting vaccine composition contained 2.2 μg of each saccharide, but 4.4 μg of 6B; approximately 50 to 90 μg of CRM 197 carrier protein; 0.125 mg elemental aluminum as an adjuvant (0.5 mg aluminum phosphate); approximately 4.25 mg sodium chloride; approximately 295 mcg succinate buffer; and approximately 100 mcg polysorbate 80 in a total volume of 0.5 mL.

[0567] Immunogenicity study of 24-valent Streptococcus pneumoniae polysaccharide-protein conjugate

[0568] OPA Analysis Results

[0569] To confirm whether the 24-valent polysaccharide-protein conjugate could induce a functional antibody response, an opsonophagocytic cell killing assay (OPA) was performed in three rabbits in the same manner as the 15-valent vaccine.

[0570] [Table 15]

[0571] Serotype PCV24 Prevnar 13 1 309 54 2 490 - 3 407 393 4 1290 2072 5 1516 306 6A 1817 2355 6B 2888 1614 7F 1277 952 8 279 - 9N 653 52 9V 178 324 10A 658 - 11A 675 - 12F 471 - 14 959 539 15B 371 - 17F 348 - 18C 1357 1996 19A 642 1870 19F 1521 1516 20 306 - 22F 1703 - 23F 1531 928 33F 428 -

[0572] According to the results, it was possible to produce a 24-valent vaccine containing all of serotypes 2, 9N, 17F, and 20. As can be seen from the results, in particular, antibodies to serotypes 2, 17F, etc., which were not produced when using Prevenar 13, could be produced. In addition, it was confirmed that the vaccine could induce an immune response against all the serotypes contained without significantly affecting the production of antibodies to antigens of other serotypes, despite the fact that 11 serotypes were added compared to Prevenar 13.

[0573] [INDUSTRIAL APPLICABILITY]

[0574] The immunogenic composition according to one example of the present invention can be used as a medicine.

[0575] An immunogenic composition according to one example of the present invention can be used in various therapeutic or prophylactic methods for preventing, treating, or alleviating bacterial infections, diseases, or conditions. In particular, the immunogenic composition described in the present invention can be used to prevent, treat, or alleviate Streptococcus pneumoniae infections, diseases, or conditions in a subject.

[0576] In one embodiment, a method for inducing an immune response to Streptococcus pneumoniae in a subject may be provided, comprising administering an effective dose of the immunogenic composition according to the present invention to the subject.

Claims

1. An immunogenic composition for preventing an infection, disease or condition caused by serotype 20 Streptococcus pneumoniae, comprising an effective amount of a conjugate of capsular polysaccharide from serotype 20 Streptococcus pneumoniae - CRM protein 197 and a pharmaceutically acceptable excipient, carrier or diluent, wherein said activated capsular polysaccharide, intended for conjugation with protein, has an oxidation state of 4-16 and a molecular weight of 400-800 kDa.

2. An immunogenic composition according to claim 1, wherein the conjugate has a molecular weight of 1000 to 4000 kDa.

3. The immunogenic composition of claim 1, wherein the ratio of capsular polysaccharide from serotype 20 to protein in said conjugate is from 1 to 5 (w / w).

4. The immunogenic composition of claim 1, wherein 70-90% of the conjugate has a Kd value of 0.3 or lower when measured in a CL-4B column.

5. The immunogenic composition of claim 1, wherein said immunogenic composition is a vaccine.

6. Use of an immunogenic composition according to any one of claims 1-5 for preventing an infection, disease or condition caused by serotype 20 Streptococcus pneumoniae.