Multivalent pneumococcal polysaccharide-protein conjugate composition

Abstract

To provide an immunogenic serotype 9N conjugate, and a mixed-carrier, multivalent pneumococcal conjugate composition comprising the same.SOLUTION: An immunogenic serotype 9N conjugate comprises a protein carrier conjugated to a capsular polysaccharide from Streptococcus pneumoniae serotype 9N, where the protein carrier is CRM197, and where the capsular polysaccharide from serotype 9N is conjugated to CRM197 in a state where the capsular polysaccharide from serotype 9N is activated to have a degree of oxidation of 2-19 or 5-10 and a molecular weight of 200-700 kDa.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of the filing dates of U.S. Provisional Patent Application No. 62 / 626,509, filed on February 5, 2018, and Korean Patent Application No. 10 - 2018 - 0045246, filed on April 18, 2018, and relies thereon, and the entire disclosures of these are incorporated herein by reference.

[0002] This application generally relates to mixed - carrier multivalent pneumococcal conjugate compositions, vaccines containing the same, and methods of use for the prevention of Streptococcus pneumoniae infections or diseases in subjects with these compositions and vaccines.

Background Art

[0003] Streptococcus pneumoniae is a gram - positive lance - shaped facultative anaerobic bacterium with over 90 known serotypes. Most serotypes of S. pneumoniae have been shown to cause disease, and 23 of the most common serotypes account for approximately 90% of invasive diseases worldwide. Serotypes are classified based on the serological reaction of the capsular polysaccharide, which is the most important pathogenic factor for pneumococci. Capsular polysaccharides are T - cell - independent antigens that induce antibody production in the absence of T - helper cells. T - cell - independent antigens generally induce antibodies with low affinity and short - lived immune responses with little to no immunological memory.

[0004] Initial pneumococcal vaccines contained combinations of capsular polysaccharides from different serotypes. These vaccines could confer immunity against S. pneumoniae in patients with a developed or healthy immune system, but were not effective in infants lacking a developed immune system and in elderly subjects who often had a reduced immune function. In particular, in infants and elderly subjects who are at a higher risk of developing S. pneumoniae infections, in order to improve the immune response to pneumococcal vaccines, the capsular polysaccharides were conjugated to a suitable carrier protein to produce pneumococcal conjugate vaccines. Conjugation to a suitable carrier protein changes the capsular polysaccharide from a T cell-independent antigen to a T cell-dependent antigen. Thus, T helper cells are involved in the immune response to the conjugated capsular polysaccharide, which aids in inducing a stronger and more rapid immune response upon re-exposure to the capsular polysaccharide.

[0005] There are at least two approaches for developing pneumococcal conjugate vaccines: the single carrier approach and the mixed carrier approach. The immunogenicity of different capsular polysaccharide conjugates can vary depending on the pneumococcal serotype and carrier protein used. In the single carrier approach, capsular polysaccharides from different serotypes are conjugated to a single protein carrier. The vaccines in Pfizer's PREVNAR series are CRM, a non-toxic variant of diphtheria toxoid with a single amino acid substitution of glycine to glutamic acid 197 This is an example of the single carrier approach where different capsular polysaccharides are conjugated to a protein carrier. The 7-valent PREVNAR vaccine (PREVNAR) was first approved in 2000 and contains capsular polysaccharides from the 7 most prevalent serotypes: 4, 6B, 9V, 14, 18C, 19F, and 23F. The 13-valent vaccine PREVNAR13 added serotypes 1, 5, 7F, 3, 6A, and 19A to the CRM 197 protein carrier. The protein carrier CRM, which is the single carrier used in the PREVNAR vaccines 197 has never been used as part of a mixed carrier system in pneumococcal conjugate vaccines.

[0006] The second pneumococcal vaccine approach is the mixed carrier approach. In the mixed carrier approach, instead of using a single protein carrier, two or more protein carriers are used, and the capsular polysaccharides from specific serotypes are conjugated to the first protein carrier, and the capsular polysaccharides from different serotypes are conjugated to at least a second different protein carrier. For example, GlaxoSmithKline developed SYNFLORIX, a 10-valent (serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F) mixed carrier pneumococcal conjugate vaccine that uses Haemophilus influenzae protein D, tetanus toxoid, and diphtheria toxoid as protein carriers. In SYNFLORIX, serotypes 1, 4, 5, 6B, 7F, 9V, 14, and 23F are conjugated to protein D, serotype 18C is conjugated to tetanus toxoid, and serotype 19F is conjugated to diphtheria toxoid [2]. Serotype 3 was partially removed from the 11-valent precursor to SYNFLORIX because it did not show serotype-specific efficacy in the acute otitis media trial [1]. Another group, Aventis Pasteur, developed an 11-valent (serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F) mixed carrier pneumococcal conjugate vaccine using diphtheria toxoid and tetanus toxoid as protein carriers [3]. Capsular polysaccharides from serotypes 3, 9V, 14, and 18C can induce a better response when conjugated to diphtheria toxoid than when conjugated to tetanus toxoid [6]. Thus, serotypes 3, 6B, 14, and 18C were conjugated to diphtheria toxin, and serotypes 1, 4, 5, 7F, 9V, 19F, and 23F were conjugated to tetanus toxoid. The development of this mixed carrier pneumococcal vaccine was discontinued, partly due to technical reasons and the potential for reduced responses when co-administered with acellular pertussis vaccines [3]. Recently, serotypes 5 and 1 were reported as having one of the lowest OPA titers observed among all 13 serotypes of PREVNAR for which there was a correlation between IgG titer and OPA activity [4].In addition, in serotype 3, it was suggested that much higher serum IgG concentrations are required for protection [5].

Summary of the Invention

[0007] This application provides a novel and improved mixed carrier multivalent pneumococcal conjugate composition and a vaccine containing the same. In one aspect, the mixed carrier multivalent pneumococcal conjugate composition comprises 21 different pneumococcal capsular polysaccharide-protein conjugates, each pneumococcal capsular polysaccharide-protein conjugate comprising a protein carrier conjugated to a capsular polysaccharide derived from a different serotype of Streptococcus pneumoniae, the Streptococcus pneumoniae serotypes being selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and the protein carrier being CRM 197 or tetanus toxoid, four of the capsular polysaccharides being conjugated to tetanus toxoid and the remaining capsular polysaccharides being conjugated to CRM 197 Of the four capsular polysaccharides conjugated to tetanus toxoid, two are selected from the group consisting of serotypes 1, 3, and 5, and the remaining two capsular polysaccharides are serotypes 15B and 22F.

[0008] In one embodiment of the mixed carrier 21-valent pneumococcal conjugate composition, the capsular polysaccharides derived from serotypes 1, 5, 15B and 22F are conjugated to tetanus toxoid, and the capsular polysaccharides derived from serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, and 33F are conjugated to CRM 197 therein.

[0009] In another embodiment of the mixed carrier 21-valent pneumococcal conjugate composition, the capsular polysaccharides derived from serotypes 1, 3, 15B and 22F are conjugated to tetanus toxoid, and the capsular polysaccharides derived from serotypes 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, and 33F are CRM197 complexed with 。

[0010] In yet another embodiment of the mixed carrier 21-valent pneumococcal conjugate composition, the capsular polysaccharides derived from serotypes 3, 5, 15B, and 22F are complexed with tetanus toxoid, and the capsular polysaccharides derived from serotypes 1, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, and 33F are complexed with CRM 197 complexed with

[0011] In another aspect, the present application is a mixed carrier multivalent pneumococcal conjugate composition comprising 21 different pneumococcal capsular polysaccharide-protein conjugates, each pneumococcal capsular polysaccharide-protein conjugate comprising a protein carrier complexed with a capsular polysaccharide derived from a different serotype of Streptococcus pneumoniae, the Streptococcus pneumoniae serotypes being selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, the protein carrier being CRM 197 or tetanus toxoid, three of the capsular polysaccharides being complexed with tetanus toxoid and the remaining capsular polysaccharides being complexed with CRM 197 complexed with, and the three capsular polysaccharides complexed with tetanus toxoid being selected from the group consisting of serotypes 1, 3, 5, 15B, and 22F, to provide a mixed carrier multivalent pneumococcal conjugate composition. In certain embodiments, two of the three capsular polysaccharides complexed with tetanus toxoid are selected from the group consisting of serotypes 1, 3, and 5, and the remaining capsular polysaccharides complexed with tetanus toxoid are serotypes 15B and 22F.

[0012] In some embodiments, the mixed carrier multivalent pneumococcal conjugate composition further comprises an adjuvant such as an aluminum-based adjuvant including, but not limited to, aluminum phosphate, aluminum sulfate, and aluminum hydroxide.

[0013] Another aspect is directed to the use of the mixed carrier 21-valent pneumococcal conjugate composition as a vaccine.

[0014] Yet another aspect is directed to a vaccine comprising a mixed carrier 21-valent pneumococcal conjugate composition and a pharmaceutically acceptable excipient.

[0015] Yet another aspect is directed to a method for the prevention of Streptococcus pneumoniae infection or disease in a subject such as a human, the method comprising administering a prophylactically effective amount of a mixed carrier 21-valent pneumococcal conjugate composition or a vaccine comprising the same to the subject.

[0016] In certain embodiments, the subject is a human at least 50 years of age and the disease is pneumonia or invasive pneumococcal disease (IPD).

[0017] In other embodiments, the subject is a human at least 6 weeks of age and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM). In some embodiments, the human subject is between 6 weeks and 5 years of age. In other embodiments, the human subject is between 2 and 15 months of age or between 6 and 17 years of age.

[0018] In certain embodiments, the mixed carrier 21-valent pneumococcal conjugate composition or vaccine is administered by intramuscular injection. In certain embodiments, the mixed carrier 21-valent pneumococcal conjugate composition or vaccine is administered as part of an immunization series.

[0019] Yet another aspect is an immunogenic conjugate of Streptococcus pneumoniae serotype 9N comprising a capsular polysaccharide from Streptococcus pneumoniae serotype 9N; and a carrier protein conjugated to the capsular polysaccharide, wherein the carrier protein is CRM 197Targeting immunogenic conjugates. In certain embodiments of immunogenic serotype 9N conjugates, mixed carrier multivalent pneumococcal conjugate compositions and vaccines (and their methods / uses), the serotype 9N polysaccharide forms a conjugate in an activated state having an oxidation degree of 2-19 or 5-10 and a molecular weight of 200-700 kDa so as to bind to CRM 197 optionally. In certain embodiments of immunogenic serotype 9N conjugates, mixed carrier multivalent pneumococcal conjugate compositions and vaccines (and their methods / uses), the immunogenic serotype 9N conjugate can have a molecular weight of 500-4,000 kDa.

[0020] In certain embodiments of immunogenic serotype 9N conjugates, mixed carrier multivalent pneumococcal conjugate compositions and vaccines (and their methods / uses), the ratio of serotype 9N capsular polysaccharide to carrier protein in the serotype 9N immunogenic conjugate is 0.1-5 (w / w). In certain embodiments, the ratio is 0.5-2.5.

[0021] In certain embodiments of immunogenic serotype 9N conjugates, mixed carrier multivalent pneumococcal conjugate compositions and vaccines (and their methods / uses), 15-60% of the immunogenic serotype 9N conjugate can have a K of 0.3 or less in a CL-4B column d In certain embodiments of immunogenic serotype 9N conjugates, mixed carrier multivalent pneumococcal conjugate compositions and vaccines (and their methods / uses), the immunogenic serotype 9N conjugate is prepared using serotype 9N polysaccharide activated to achieve an oxidation degree of 2-19. In certain embodiments, the immunogenic serotype 9N conjugate is prepared using serotype 9N polysaccharide activated to achieve an oxidation degree of 5-10.

[0022]

[0023] ​In certain embodiments of the immunogenic serotype 9N conjugate, the mixed carrier multivalent pneumococcal conjugate composition and vaccine (as well as their methods / uses), the Streptococcus pneumoniae serotype 9N polysaccharide is CRM by adding 0.02 - 0.19 μg of periodate per μg of polysaccharide. 197 When complexed with, the complex has a molecular weight of 500 - 4,000 kDa, a molecular weight distribution of 15 - 60% (K d ≤0.3) and a polysaccharide / protein ratio of 0.5 - 2.5.

[0024] In yet another aspect, the disclosure also provides a method for preparing an immunogenic conjugate of Streptococcus pneumoniae serotype 9N, comprising: (a) lysing bacterial cells that produce the capsular polysaccharide of Streptococcus pneumoniae serotype 9N by fermenting it; (b) purifying the capsular polysaccharide of Streptococcus pneumoniae serotype 9N from the lysed cells; (c) activating the capsular polysaccharide of Streptococcus pneumoniae serotype 9N by reacting it with an oxidizing agent to achieve an oxidation degree of 2 - 19 or 5 - 10; (d) forming a complex of the capsular polysaccharide of Streptococcus pneumoniae serotype 9N bound to CRM 197 by mixing the activated polysaccharide with CRM 197 . The method is also provided.

[0025] In certain embodiments, the CRM mixed in step (d) 197 can be reacted with a reducing agent to form a complex with the activated capsular polysaccharide of Streptococcus pneumoniae serotype 9N. In certain embodiments, in step (c), 0.02 - 0.19 μg of periodate can be reacted with 1 μg of the capsular polysaccharide of Streptococcus pneumoniae serotype 9N at 20 - 25 °C for 15 - 20 hours.

[0026] In certain embodiments, the capsular polysaccharide of Streptococcus pneumoniae serotype 9N reacted with an oxidizing agent in step (c) may have a molecular weight of 400-900 kDa. In certain embodiments, the activated capsular polysaccharide of Streptococcus pneumoniae serotype 9N mixed with CRM 197 in step (d) may have a molecular weight of 200-700 kDa. In certain embodiments, the immunogenic complex of Streptococcus pneumoniae serotype 9N may have a molecular weight of 500-4,000 kDa. In certain embodiments, the initial loading ratio of CRM 197 to the activated capsular polysaccharide of serotype 9N (carrier CRM 197 : capsular polysaccharide) may be 0.5-2.5:1. In certain embodiments, at least 15-60% of the immunogenic complex may have a K d of 0.3 or less as measured by a CL-4B column.

[0027] In certain embodiments, when the polysaccharide of Streptococcus pneumoniae serotype 9N of the present disclosure is complexed with CRM by adding 0.02-0.19 μg of periodate per μg of sugar, the immunogenic complex has a molecular weight of 500-4,000 kDa, a molecular weight distribution of 15-60% (K 197 ≤0.3) as measured by a CL-4B column, and a CRM d / polysaccharide ratio of 0.5-2.5. 197

[0028] The foregoing and other objects, features, and advantages of the mixed carrier 21-valent pneumococcal conjugate composition will become more apparent from the following detailed description.

[0029] Definitions To facilitate a better understanding of the present disclosure, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth throughout the specification.

[0030] ​As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods and / or steps of the kind described herein and / or made apparent to one of ordinary skill in the art by reading the present disclosure, and / or the like.

[0031] Administer: As used herein, "administering" a composition to a subject means giving, applying, or contacting the composition to the subject. Administration can be accomplished by any of a number of routes, such as, for example, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal, and intradermal.

[0032] Approximately: As used herein, the term "approximately" or "about", when applied to one or more values of interest, refers to a value similar to the recited reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the recited reference value, unless otherwise stated or otherwise apparent from the context (except where such number would exceed 100% of the possible value).

[0033] Complex: As used herein and understood in the appropriate context, the term "complex(es)" or "glycoconjugate(s)" refers to Streptococcus pneumoniae polysaccharides conjugated to a carrier protein using any covalent or non-covalent bioconjugation strategy.

[0034] Degree of oxidation: As used herein, the term "degree of oxidation" (DO) refers to the number of sugar repeat units per aldehyde group that occur when a purified or size-adjusted glycoconjugate is activated with an oxidizing agent. The degree of oxidation of a glycoconjugate can be determined using conventional methods known to those of ordinary skill in the art. refers to the number of sugar repeat units per aldehyde group that occur when a purified or size-adjusted glycoconjugate is activated with an oxidizing agent. The degree of oxidation of a glycoconjugate can be determined using conventional methods known to those of ordinary skill in the art.

[0035] Excipient: As used herein, the term "excipient" refers to a non-therapeutic agent that may be included in a composition, for example, to provide or contribute to a desired consistency or stabilizing effect.

[0036] Mixed carrier: As used herein, a mixed carrier pneumococcal conjugate composition refers to a pneumococcal conjugate composition having multiple types of protein carriers.

[0037] Polyvalent: As used herein, the term "polyvalent" refers to a pneumococcal conjugate composition having pneumococcal capsular polysaccharides derived from multiple Streptococcus pneumoniae serotypes.

[0038] Mixed carrier 21-valent pneumococcal conjugate composition: As used herein, the term "mixed carrier 21-valent pneumococcal conjugate composition(s)" is a composition comprising or consisting of 21 different pneumococcal capsular polysaccharide-protein conjugates, wherein each pneumococcal capsular polysaccharide-protein conjugate comprises a protein carrier conjugated to a capsular polysaccharide derived from a different serotype of Streptococcus pneumoniae, and the Streptococcus pneumoniae serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, and the protein carrier is CRM 197 or tetanus toxoid, 1) three of the capsular polysaccharides are conjugated to tetanus toxoid, and the remaining capsular polysaccharides are conjugated to CRM 197 and two of the three capsular polysaccharides conjugated to tetanus toxoid are selected from the group consisting of serotypes 1, 3, and 5, and the remaining capsular polysaccharide is serotype 15B or 22F, or 2) four of the capsular polysaccharides are conjugated to tetanus toxoid, and the remaining capsular polysaccharides are conjugated to CRM 197is complexed to, and two of the four capsular polysaccharides complexed to tetanus toxoid are selected from the group consisting of serotypes 1, 3, and 5, and the remaining two capsular polysaccharides are serotype 15B or 22F, referring to a composition. In some embodiments, the capsular polysaccharides from serotypes 1, 5, 15B, and 22F are complexed to tetanus toxoid, and the remaining capsular polysaccharides are CRM 197 is complexed to. In another embodiment, the capsular polysaccharides from serotypes 1, 3, 15B, and 22F are complexed to tetanus toxoid, and the capsular polysaccharides from the remaining serotypes are CRM 197 is complexed to. In yet another embodiment, the capsular polysaccharides from serotypes 3, 5, 15B, and 22F are complexed to tetanus toxoid, and the remaining capsular polysaccharides are CRM 197 is complexed to.

[0039] Molecular weight: Unless otherwise specified, as used herein, the term "molecular weight" of a capsular polysaccharide or a capsular polysaccharide-carrier protein complex refers to the average molecular weight calculated by size exclusion chromatography (SEC) combined with multi-angle laser light scattering (MALLS).

[0040] Pharmaceutically acceptable excipients: The pharmaceutically acceptable excipients useful in the present disclosure are conventional. Remington’s Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 15 th Edition (1975) describes compositions and formulations suitable for the pharmaceutical delivery of vaccines, and one or more therapeutic compositions including additional pharmaceutical agents. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, mono It contains glycerol stearate, talc, sodium chloride, dry skim milk, glycerol, propylene, glycol, water, ethanol, etc. Generally, the nature of the excipient depends on the specific dosage form used. For example, parenteral formulations usually include injections containing pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, buffers, aqueous dextrose, glycerol, etc. as vehicles. In the case of solid compositions (e.g., powders, pills, tablets, or capsule forms), conventional non-toxic solid excipients may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions administered may contain trace amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, surfactants, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0041] Prophylactically effective amount: As defined herein, the term "prophylactically effective amount" or "prophylactically effective dosage" refers to the amount or dosage necessary to delay the onset of one or more symptoms caused by infection with Streptococcus pneumoniae and / or to induce an immune response sufficient to reduce the frequency and / or severity thereof.

[0042] Prophylaxis: As used herein, the term "prophylaxis" refers to the avoidance, delay in the onset, and / or reduction in the frequency and / or severity of the symptoms of a particular disease, disorder, or condition (e.g., an infection by Streptococcus pneumoniae). In some embodiments, prophylaxis is evaluated on a population basis such that a drug is considered to provide prophylaxis against a particular disease, disorder, or condition if a statistically significant reduction in the onset, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population that is susceptible to the disease, disorder, or condition.

[0043] Subject: As used herein, the term "subject" means any mammal, including mice, rabbits, and humans. In certain embodiments, the subject is an adult, adolescent, or infant. In some embodiments, the terms "individual" or "patient" are used and are intended to be interchangeable with "subject."

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The following description of the disclosed embodiments and examples is illustrative only and is in no way intended to limit the invention, its application, or uses.

[0045] This application provides novel and improved mixed carrier multivalent pneumococcal conjugate compositions and vaccines containing the same. The protein carrier CRM 197 has been previously used in single carrier pneumococcal conjugate vaccines, but this application describes the use of CRM 197 in mixed carrier pneumococcal conjugate vaccines. In particular, this application describes the use of CRM 197 and tetanus toxoid as carrier proteins for specific pneumococcal serotypes in multivalent pneumococcal conjugate compositions and vaccines.

[0046] As discussed above, the immunogenicity of different capsular polysaccharide conjugates can vary depending on the pneumococcal serotype and carrier protein used. This application describes the successful conjugation of serotype 3 to tetanus toxoid as part of a mixed carrier vaccine, despite previous findings [6] that immunogenicity is higher when serotype 3 is conjugated to diphtheria toxoid rather than tetanus toxoid. This application also describes the successful conjugation of serotypes 1, 5, 15B, and 22F to tetanus toxoid as part of a mixed carrier vaccine. Also, the antibody response to serotype 3 conjugated to tetanus toxoid in a mixed carrier multivalent, e.g., 21-valent, pneumococcal conjugate composition was compared to the antibody response to serotype 3 when it was CRM 197It also discloses the unexpected finding that it was approximately 4.5 times higher than when complexed.

[0047] Furthermore, this unexpected finding was not limited to serotype 3 and was also observed for other serotypes complexed to tetanus toxoid in the mixed carrier multivalent pneumococcal conjugate composition. For example, as shown in the examples, the complexation of serotypes 1 and 5 or 3 and 5 to tetanus toxoid in the mixed carrier pneumococcal conjugate composition, where the remaining serotypes are CRM 197 complexed thereto (e.g., PCV21(1 / 5 / 15B / 22F)-TT and PCV21(3 / 5 / 15B / 22F)-TT), consistently induced a significantly enhanced antibody response to the serotypes complexed to tetanus toxoid compared to the antibody response (IgG response or MOPA titer) to the same serotypes complexed to CRM 197 in the single carrier, pneumococcal conjugate composition (PREVNAR13).

[0048] Tetanus toxoid is significantly larger than CRM 197 . Therefore, complexing three or four of serotypes 1, 3, 5, 15B, and 22F to tetanus toxoid as part of a mixed carrier vaccine results in a decreased polysaccharide to carrier ("PS / C") ratio for those serotypes complexed to tetanus toxoid compared to the PS / C ratio of those same serotypes complexed to a single carrier smaller than tetanus toxoid, such as CRM 197 . Thus, the mixed carrier approach described in this application can be used to lower the PS / C ratio for one or more of serotypes 1, 3, 5, 15B, or 22F.

[0049] The 21-valent pneumococcal conjugate composition described in the present application also includes pneumococcal serotypes not currently covered by the three pneumococcal conjugate vaccines available on the world market today: PREVNAR (called Prevenar in some countries), SYNFLORIX, and PREVNAR13. Diseases caused by pneumococcal serotypes not currently covered are increasing, due in part to the emergence of antimicrobial resistance, the increasing number of immunocompromised patients, and the lack of immune pressure. For example, none of the currently available pneumococcal conjugate vaccines include serotype 9N. Also, none of the currently available pneumococcal conjugate vaccines include serotypes 8, 10A, 11A, 12F, 15B, 22F, and 33F. The present disclosure demonstrates the successful implementation of a conjugate (tetanus toxoid and CRM 197 ) for serotypes 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F into a pneumococcal conjugate vaccine, and that serotype 9N induces an antibody reactivity approximately 40 - 50 times higher than PREVNAR13.

[0050] Pneumococcal polysaccharide serotype 9N Serotype 9N polysaccharide can be obtained directly from bacteria by using isolation procedures known to those skilled in the art (including, but not limited to, the method disclosed in U.S. Patent Application Publication No. 2006 / 0228380). Also, the polysaccharide can be manufactured using a synthetic protocol.

[0051] Streptococcus pneumoniae strains of serotype 9N can be obtained from established culture repositories (e.g., the Streptococcal Reference Laboratory of the Centers for Disease Control and Prevention (Atlanta, Georgia)) or clinical specimens.

[0052] The bacterial cells typically grow in a medium such as a soybean-based medium. Following fermentation of the bacterial cells that produce the capsular polysaccharide of Streptococcus pneumoniae serotype 9N, the bacterial cells are lysed to produce a cell lysate. Subsequently, the serotype 9N polysaccharide can be isolated from the cell lysate using purification techniques known in the art, including, but not limited to, centrifugation, depth filtration, precipitation, ultrafiltration, treatment with activated carbon, diafiltration, and / or column chromatography (the methods disclosed in U.S. Patent Application Publication No. 2006 / 0228380). The purified serotype 9N capsular polysaccharide can be used in the preparation of immunogenic conjugates. The serotype 9N capsular polysaccharide obtained by purifying the serotype 9N polysaccharide from Streptococcus pneumoniae lysate and optionally sizing the purified polysaccharide can be characterized by different parameters, including, for example, the molecular weight (MW) of the serotype 9N capsular polysaccharide.

[0053] In certain embodiments, the purified polysaccharide purified from Streptococcus pneumoniae serotype 9N prior to conjugation has a molecular weight of 5 to 5,000 kDa. In certain embodiments, the serotype 9N capsular polysaccharide prior to conjugation has a molecular weight of 50 to 1,000 kDa. In certain embodiments, the serotype 9N capsular polysaccharide prior to conjugation has a molecular weight of 70 to 900 kDa. In certain embodiments, the serotype 9N capsular polysaccharide prior to conjugation has a molecular weight of 100 to 800 kDa. In certain embodiments, the purified serotype 9N capsular polysaccharide can be activated prior to conjugation to have a molecular weight in the range of 50 to 800 kDa, 80 to 780 kDa, 100 to 770 kDa, 120 to 760 kDa, 140 to 750 kDa, 150 to 740 kDa, 160 to 730 kDa, 170 to 735 kDa, 180 to 720 kDa, 190 to 710 kDa, 200 to 700 kDa, 220 to 690 kDa, 240 to 680 kDa, 260 to 670 kDa, 270 to 660 kDa, or a similar molecular weight range. Integers within any of the above ranges are contemplated as embodiments of the present disclosure.

[0054] The activated serotype 9N polysaccharide can be characterized by its degree of oxidation and molecular weight. In certain embodiments, the activated serotype 9N polysaccharide can have a degree of oxidation of 0.5 - 25, 0.6 - 23, 0.8 - 21, 1 - 20.8, 1.1 - 20.5, 1.2 - 20.3, 1.3 - 20, 1.4 - 19.5, 1.5 - 19.3, 1.6 - 19.2, 1.7 - 19.1, 2 - 19, 3 - 18, 4 - 15, or 5 - 10.

[0055] The polysaccharide can be slightly reduced in size during normal purification procedures. Also, as described in the present disclosure, the polysaccharide can be subjected to size adjustment prior to complexation. The above molecular weight ranges refer to those of the purified polysaccharide after the final size adjustment step prior to complexation (e.g., after purification, hydrolysis, and activation).

[0056] Mixed carrier polyvalent pneumococcal conjugate composition and method for producing the same The present disclosure provides a mixed carrier polyvalent pneumococcal conjugate composition comprising or consisting of 21 different pneumococcal capsular polysaccharide - protein conjugates, wherein each pneumococcal capsular polysaccharide - protein conjugate comprises a protein carrier conjugated to a capsular polysaccharide derived from a different serotype of Streptococcus pneumoniae, and the Streptococcus pneumoniae serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, and the protein carrier is CRM 197 or tetanus toxoid, wherein 3 - 4 of the capsular polysaccharides are conjugated to tetanus toxoid and the remaining capsular polysaccharides are conjugated to CRM 197 There is provided a mixed carrier polyvalent pneumococcal conjugate composition, wherein the 3 - 4 capsular polysaccharides conjugated to tetanus toxoid are selected from the group consisting of serotypes 1, 3, 5, 15B, and 22F. In certain embodiments, 3 of the capsular polysaccharides are conjugated to tetanus toxoid and the remaining capsular polysaccharides are conjugated to CRM 197is conjugated. In certain embodiments, four of the capsular polysaccharides are conjugated to tetanus toxoid and the remaining capsular polysaccharides are CRM 197 is conjugated.

[0057] In one aspect, the disclosure provides a mixed carrier multivalent pneumococcal conjugate composition comprising or consisting of 21 different pneumococcal capsular polysaccharide-protein conjugates, each pneumococcal capsular polysaccharide-protein conjugate comprising a protein carrier conjugated to a capsular polysaccharide from a different serotype of Streptococcus pneumoniae, the Streptococcus pneumoniae serotypes being 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, and the protein carrier being CRM 197 or tetanus toxoid, four of the capsular polysaccharides being conjugated to tetanus toxoid and the remaining capsular polysaccharides being CRM 197 is conjugated, two of the four capsular polysaccharides conjugated to tetanus toxoid being selected from the group consisting of serotypes 1, 3, and 5 and the remaining two capsular polysaccharides being serotypes 15B and 22F.

[0058] In one embodiment, the capsular polysaccharides from serotypes 1, 5, 15B, and 22F are conjugated to tetanus toxoid and the remaining capsular polysaccharides are CRM 197 is conjugated. In another embodiment, the capsular polysaccharides from serotypes 1, 3, 15B, and 22F are conjugated to tetanus toxoid and the remaining capsular polysaccharides are CRM 197 is conjugated. In yet another embodiment, the capsular polysaccharides from serotypes 3, 5, 15B, and 22F are conjugated to tetanus toxoid and the remaining capsular polysaccharides are CRM 197 is conjugated.

[0059] In a polysaccharide-protein conjugate vaccine, the carrier protein is conjugated to the polysaccharide antigen, mainly to assist in enhancing the immune response (e.g., antibody response) against the polysaccharide antigen. The carrier protein is preferably a non-toxic protein. The carrier protein should follow the conjugation with pneumococcal polysaccharide using standard conjugation procedures, as discussed in more detail below. The carrier proteins used in the mixed carrier 21-valent pneumococcal conjugate composition are tetanus toxoid (TT) and CRM 197 and each of these has been used in the design of pneumococcal conjugate vaccines, but has never been used in the same mixed carrier vaccine.

[0060] CRM 197 is a non-toxic variant of diphtheria toxin (i.e., toxoid) that retains the immunological properties of the wild-type diphtheria toxin. CRM 197 differs from the wild-type diphtheria toxin by one base in the structural gene, with one amino acid substitution from glutamic acid to glycine. CRM 197 is typically isolated from a culture of Corynebacterium diphtheria strain C7(β197) grown in a medium based on casamino acids and yeast extract. CRM 197 can be purified by ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography. Alternatively, CRM 197 can be prepared recombinantly according to U.S. Patent No. 5,614,382, which is incorporated herein by reference in its entirety. CRM 197 has been used in the design of pneumococcal conjugate vaccines, but has never been used as part of a mixed carrier vaccine.

[0061] Tetanus toxoid is prepared and used worldwide for large-scale immunization against tetanus (or lockjaw) caused by Clostridium tetani. Tetanus toxoid is also used alone and in combination with diphtheria and / or pertussis vaccines. The parent protein, tetanus toxin, is generally obtained from cultures of Clostridium tetani. Tetanus toxin is a protein of approximately 150 kDa and consists of two subunits (approximately 100 kDa and approximately 50 kDa) linked by disulfide bonds. The toxin is typically detoxified with formaldehyde and can be purified from the culture filtrate using known methods such as ammonium sulfate precipitation (see, for example, [7], [8]) or chromatography techniques (as disclosed, for example, in WO1996 / 025425). Tetanus toxin can also be inactivated by recombinant genetic means.

[0062] Tetanus toxoid has also been used as a carrier protein in other vaccines, including pneumococcal conjugate vaccines. However, it is novel to use tetanus toxin in combination with CRM 197 in a mixed carrier pneumococcal conjugate vaccine. The art also teaches away from conjugating serotype 3 to tetanus toxoid in a mixed carrier pneumococcal conjugate vaccine because serotype 3 has been shown to be more immunogenic when conjugated to diphtheria toxoid compared to tetanus toxoid [6].

[0063] The pneumococcal capsular polysaccharides used in the compositions and vaccines described herein, which comprise capsular polysaccharides derived from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, can be prepared from Streptococcus pneumoniae using any available technique, including standard techniques known to those of skill in the art, including, for example, those disclosed in WO2006 / 110381, WO2008 / 118752, WO2006 / 110352, and U.S. Patent Application Publication Nos. 2006 / 0228380, 2006 / 0228381, 2007 / 0184071, 2007 / 0184072, 2007 / 0231340, 2008 / 0102498, and 2008 / 0286838 (all of which are incorporated by reference in their entirety). For example, each serotype of pneumococcal capsular polysaccharide can be grown in a culture medium (e.g., a soy-based medium). The cells are lysed, and the individual polysaccharides can be purified from the lysate by centrifugation, precipitation, ultrafiltration, and / or column chromatography. Also, pneumococcal capsular polysaccharides can be produced using synthetic protocols.

[0064] The capsular polysaccharides of Streptococcus pneumoniae contain repeating oligosaccharide units that can contain up to eight sugar residues. The capsular polysaccharide antigen can be the full-length polysaccharide or can be of reduced size (e.g., a single oligosaccharide unit or shorter than the native-length sugar chain of the repeating oligosaccharide units). The size of the capsular polysaccharide can be reduced by acid hydrolysis treatment, hydrogen peroxide treatment, sizing by generating oligosaccharide fragments by high-pressure homogenizer followed optionally by hydrogen peroxide treatment, or by various methods known in the art such as microfluidization.

[0065] Each pneumococcal conjugate of a serotype can be prepared by conjugating the capsular polysaccharide of each serotype to a carrier protein. Different pneumococcal conjugates can be formulated into a composition containing a single dosage formulation.

[0066] To prepare the polysaccharide-protein complex, the capsular polysaccharide prepared from each pneumococcal serotype can be chemically activated such that the capsular polysaccharide can react with the carrier protein. Once activated, each capsular polysaccharide can be separately complexed to the carrier protein to form the glycoconjugate. Chemical activation of the polysaccharide and subsequent complexation to the carrier protein can be achieved by conventional methods. For example, the vicinal hydroxyl groups at the termini of the capsular polysaccharide can be oxidized to aldehyde groups by an oxidizing agent such as periodate (sodium periodate, potassium periodate, or periodic acid), as disclosed in, for example, U.S. Pat. Nos. 4,365,170, 4,673,574, and 4,902,506, which are hereby incorporated by reference in their entirety. Periodate randomly oxidizes the vicinal hydroxyl groups of the carbohydrate to form reactive aldehyde groups and causes cleavage of the C-C bond. The term "periodate" includes both periodate and periodic acid. This term also includes both metaperiodate (IO 4- ) and orthoperiodate (IO 65- ). The term "periodate" also includes the various salts of periodate, including sodium periodate and potassium periodate. In certain embodiments , the polysaccharide can be oxidized in the presence of sodium metaperiodate.

[0067] In certain embodiments, the periodate can be used in an amount of about 0.03 to 0.17 μg per μg of polysaccharide. In certain embodiments, the periodate can be used in an amount of about 0.025 to 0.18 μg or about 0.02 to 0.19 μg per μg of polysaccharide. The polysaccharide can be activated as desired within the above ranges. Outside of that range, the effect may be insufficient.

[0068] The polysaccharide can also be activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide is then attached directly or via a spacer or linker group to an amino group on the carrier protein.

[0069] For example, the spacer can be cystamine or cysteamine to provide a thiolated polysaccharide that can be coupled to a carrier via a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyloxy] succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g., iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfosuccinimidyl-SIAB), N-succinimidyl iodoacetate (SIA) or succinimidyl 3-[bromoacetamido] propionate (SBAP)). Preferably, the cyanate ester (optionally produced by COAP chemistry) is coupled to hexanediamine or adipic acid dihydrazide (AOH), and the amino-derivatized glycoside is complexed to the carrier protein using carbodiimide (e.g., EDAC or EDC) chemistry via a carboxyl group on the protein carrier. Such complexes are described, for example, in WO93 / 15760, WO95 / 08348 and WO96 / 129094, all of which are hereby incorporated by reference in their entirety.

[0070] The complexation of an activated capsular polysaccharide and a carrier protein can be achieved, for example, by reductive amination, as described in, for example, U.S. Patent Application Publication Nos. 2006 / 0228380, 2007 / 0231340, 2007 / 0184071 and 2007 / 0184072, WO2006 / 110381, WO2008 / 079653, and WO2008 / 143709 (all of which are incorporated by reference in their entirety). For example, an activated capsular polysaccharide and a carrier protein can be reacted with a reducing agent to form a complex. Suitable reducing agents include borohydrides such as sodium cyanoborohydride, borane-pyridine, sodium triacetoxyborohydride, sodium borohydride, or a borohydride ion exchange resin. At the end of the reduction reaction, unreacted aldehyde groups may remain in the complex. The unreacted aldehyde groups can be capped using a suitable capping agent such as sodium borohydride (NaBH 4 ). In one embodiment, the reduction reaction is carried out in an aqueous solvent. In another embodiment, the reaction is carried out in an aprotic solvent. In an embodiment, the reduction reaction is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent. Other possible reducing agents include amine-boranes such as pyridine-borane, 2-picoline-borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine-borane (PEMB), but are not limited thereto.

[0071] The activated capsular polysaccharide can be complexed directly to the carrier protein or indirectly through the use of a spacer or linker, such as a bifunctional linker. The linker can optionally be heterobifunctional or homobifunctional and has, for example, a reactive amino group and a reactive carboxylic acid group, two reactive amino groups or two reactive carboxylic acid groups.

[0072] Other suitable techniques for complexation include, for example, the use of carbodiimides, hydrazides, active esters, norbornanes, p-nitrobenzoic acid, N-hydroxysuccinimide, S--NHS, EDC, TSTU as described in International Patent Application Publication WO98 / 42721 (incorporated by reference in its entirety). Complexation may involve a carbonyl linker that can be formed by the reaction of a free hydroxyl group of the saccharide with 1,1'-carbonyldiimidazole (CDl) (see Bethell et al. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al. (1981) J. Chromatogr. 218:509-518) followed by reaction with a protein to form a carbamate bond. This can include reduction to the primary hydroxyl group at the anomeric terminus, optional protection / deprotection of the primary hydroxyl group, formation of a CDI carbamate intermediate by reaction of the primary hydroxyl group with CDI, and coupling of the CDl carbamate intermediate with an amino group on the protein.

[0073] The ratio of polysaccharide to carrier protein for a pneumococcal conjugate vaccine is typically in the range of 0.3 - 3.0 (w / w), but can vary by serotype. The ratio can be determined by independent measurement of the amounts of protein and polysaccharide present, or by methods that provide a direct measurement of the ratio known in the art. 1 Methods including SEC-HPLC-UV / RI with H NMR spectroscopy or dual monitoring (e.g., refractive index and UV (for total substance and protein content respectively)) can profile the saccharide / protein ratio across the size distribution of the complex, similar to SEC-HPLC-MALLS or MALDI-TOF-MS.

[0074] The polysaccharide-protein complex thus obtained can be purified and concentrated by various methods. These methods include concentration / diafiltration, column chromatography, and depth filtration. The purified polysaccharide-protein complex can be combined to formulate a mixed carrier 21-valent pneumococcal conjugate composition, which can be used as a vaccine.

[0075] The formulation of the vaccine composition can be achieved using methods recognized in the art. The vaccine composition is formulated to be compatible with its intended route of administration. The individual pneumococcal capsular polysaccharide-protein complexes can be formulated with a physiologically acceptable vehicle for preparing the composition. Examples of such vehicles include, but are not limited to, water, buffered saline, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol), and dextrose solutions.

[0076] In some embodiments, the mixed carrier 21-valent pneumococcal conjugate composition further comprises an adjuvant. As used herein, "adjuvant" refers to a substance or vehicle that nonspecifically enhances the immune response to an antigen. Adjuvants can include suspensions of minerals (such as alum, aluminum salts, e.g., aluminum hydroxide, aluminum phosphate, aluminum sulfate, aluminum hydroxyphosphate sulfate, etc.) to which the antigen is adsorbed, or oil-in-water emulsions (such as Freund's incomplete adjuvant) in which the antigen solution is emulsified in a mineral oil, optionally containing heat-killed mycobacteria to further enhance antigenicity (Freund's complete adjuvant). Immunostimulatory oligonucleotides (such as those containing CpG motifs) can also be used as adjuvants (see, e.g., U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants also include biological molecules, such as lipids and costimulatory molecules. Exemplary biological adjuvants include 、 including AS04 [9], IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41BBL.

[0077] In some embodiments, the adjuvant is an aluminum-based adjuvant. Typically, a single 0.5 ml vaccine dose is formulated to contain from about 0.1 mg to 2.5 mg of an aluminum-based adjuvant. In other embodiments, a single 0.5 ml vaccine dose is formulated to contain from 0.1 mg to 2 mg, from 0.1 mg to 1 mg, from 0.1 mg to 0.5 mg, from 0.1 mg to 0.2 mg, from 0.125 mg to 2.5 mg, from 0.125 mg to 0.5 mg, from 0.125 mg to 0.2 mg or from 0.125 to 0.25 mg of an aluminum-based adjuvant. In certain embodiments, a single 0.5 ml vaccine dose is formulated to contain from about 0.125 mg to about 0.250 mg of an aluminum-based adjuvant. In certain embodiments, a single 0.5 ml vaccine dose is formulated to contain about 0.125 mg of an aluminum-based adjuvant. In certain embodiments, a single 0.5 ml vaccine dose is formulated to contain about 0.250 mg of an aluminum-based adjuvant.

[0078] In certain embodiments, the adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide.

[0079] In certain embodiments, the adjuvant is aluminum phosphate.

[0080] In some embodiments, the composition is for use as a vaccine against infections by Streptococcus pneumoniae.

[0081] Characterization of the Pneumococcal Capsular Polysaccharide-Protein Carrier Conjugate In certain embodiments, the polysaccharide-protein carrier complex can have a molecular weight of 100 to 10,000 kDa. In certain embodiments, the complex has a molecular weight of 200 to 9,000 kDa. In certain embodiments, the complex has a molecular weight of 300 to 8,000 kDa. In certain embodiments, the complex has a molecular weight of 400 to 7,000 kDa. In certain embodiments, the complex has a molecular weight of 500 to 6,000 kDa. In certain embodiments, the complex has a molecular weight of 600 to 5,000 kDa. In certain embodiments, the complex has a molecular weight of 500 to 4,000 kDa. Integers within any of the above ranges are contemplated as embodiments of the present disclosure.

[0082] When the molecular weight is within the above range, the complex can be formed stably with a high yield. Also, the proportion of free polysaccharide can be reduced. Also, excellent immunogenicity can be obtained within the above molecular weight range.

[0083] After the individual polysaccharide-protein complexes are purified, they are admixed to formulate the immunogenic composition of the present disclosure.

[0084] The serotype sugar-protein complexes of the present disclosure can be characterized by the ratio of polysaccharide to protein carrier (amount of polysaccharide / amount of protein carrier, w / w) for the protein carrier.

[0085] In certain embodiments, for each serotype, the ratio (w / w) of polysaccharide to protein carrier in the polysaccharide-protein carrier complex is 0.5 to 2.5, 0.4 to 2.3, 0.3 to 2.1, 0.24 to 2, 0.2 to 1.8, 0.18 to 1.6, 0.16 to 1.4, 0.14 to 1.2, 0.12 to 1 or 0.1 to 1 (e.g., about 0.7, about 0.8, about 0.9, 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 or about 2.5).

[0086] When the ratio of the polysaccharide to the protein carrier is within the above range, the complex can be formed stably in high yield. Also, the proportion of free polysaccharide can be reduced. Further, excellent immunogenicity can be achieved and the complex can be stably maintained without interference by other serotypes within the above range.

[0087] The complexes and immunogenic compositions of the present disclosure may contain free polysaccharides that are not covalently conjugated to the protein carrier but are present in the polysaccharide-protein carrier complex composition nevertheless. The free polysaccharides may be non-covalently associated with the polysaccharide-protein carrier complex (i.e., non-covalently bound to, adsorbed to, or entrapped within or by the polysaccharide-protein carrier complex).

[0088] In certain embodiments, the polysaccharide-protein carrier complex contains less than about 60%, about 50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% of the free polysaccharide of each serotype, based on the total amount of the polysaccharide of each serotype. In certain embodiments, the polysaccharide-protein carrier complex of each serotype contains less than about 60% of the free polysaccharide of each serotype, based on the total amount of the polysaccharide of each serotype. In certain embodiments, the polysaccharide-protein carrier complex of each serotype contains less than about 50% of the free polysaccharide of each serotype, based on the total amount of the polysaccharide of each serotype. In certain embodiments, the polysaccharide-protein carrier complex of each serotype contains less than about 40% of the free polysaccharide of each serotype, based on the total amount of the polysaccharide of each serotype. In certain embodiments, the polysaccharide-protein carrier complex of each serotype contains less than about 30% of the free polysaccharide of each serotype, based on the total amount of the polysaccharide of each serotype. In certain embodiments, the polysaccharide-protein carrier complex of each serotype contains less than about 25% of the free polysaccharide of each serotype, based on the total amount of the polysaccharide of each serotype. In certain embodiments, the polysaccharide-protein carrier complex of each serotype contains less than about 20% of the free polysaccharide of each serotype, based on the total amount of the polysaccharide of each serotype. In certain embodiments, the polysaccharide-protein carrier complex of each serotype contains less than about 15% of the free polysaccharide of each serotype, based on the total amount of the polysaccharide of each serotype. In certain embodiments, the polysaccharide-protein carrier complex of each serotype contains less than about 10% of the free polysaccharide of each serotype, based on the total amount of the polysaccharide of each serotype.

[0089] The polysaccharide-protein carrier complex of each serotype also has its molecular size distribution (K d) can be characterized by. To determine the relative molecular size distribution of the complex, a size exclusion chromatography medium (CL-4B; cross-linked agarose beads, 4%) can be used. Size exclusion chromatography (SEC) is used in a gravity-fed column to profile the molecular size distribution of the complex. Large molecules excluded from the pores in the medium elute faster than small molecules. A fraction collector is used to collect the column eluate. The fractions are tested by colorimetric analysis using a ribosome assay. K d For the determination, the column is calibrated to achieve a fraction (V 0 ; K d = 0) where the molecules are completely excluded and a fraction (V i ; K d = 1) representing the maximum retention. The fraction (V e ) reaching a particular sample property is related to K d by the equation K e = (V 0 - V i ) / (V 0 - V d ).

[0090] In certain embodiments, at least 15% of the polysaccharide-protein carrier complex of each serotype can have a K d of 0.3 or less in a CL-4B column.

[0091] In certain embodiments, at least 20% of the polysaccharide-protein carrier complex of each serotype can have a K d of 0.3 or less in a CL-4B column. In certain embodiments, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the polysaccharide-protein carrier complex of each serotype can have a K d of 0.3 or less in a CL-4B column. In certain embodiments, at least 60% of the polysaccharide-protein carrier complex of each serotype can have a K dmay have. In certain embodiments, at least 50-80% of the polysaccharide-protein carrier complexes of each serotype have a K of 0.3 or less on a CL-4B column d may have. In certain embodiments, at least 65-80% of the polysaccharide-protein carrier complexes of each serotype have a K of 0.3 or less on a CL-4B column d may have. In certain embodiments, at least 15-60% of the polysaccharide-protein complexes of each serotype have a K of 0.3 or less on a CL-4B column d may have.

[0092] Preventive methods and uses In one aspect, the present disclosure provides a vaccine comprising a mixed carrier 21-valent pneumococcal conjugate composition and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient includes at least a buffer such as succinate buffer, a salt such as sodium chloride, and / or a surfactant such as polyoxyethylene sorbitan ester (e.g., polysorbate 80). In some embodiments, three or four capsular polysaccharides from specific serotypes as described above are conjugated to tetanus toxoid, and the remaining capsular polysaccharides among 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F are conjugated to CRM 197 (21-valent).

[0093] In one embodiment, the capsular polysaccharides from serotypes 1, 5, 15B, and 22F are conjugated to tetanus toxoid, and the capsular polysaccharides from serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, and 33F are conjugated to CRM 197 (21-valent).

[0094] In another embodiment, the capsular polysaccharides derived from serotypes 1, 3, 15B, and 22F are conjugated to tetanus toxoid, and the capsular polysaccharides derived from serotypes 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, and 33F are conjugated to CRM 197 (21-valent).

[0095] In yet another embodiment, the capsular polysaccharides derived from serotypes 3, 5, 15B, and 22F are conjugated to tetanus toxoid, and the capsular polysaccharides derived from serotypes 1, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, and 33F are conjugated to CRM 197 (21-valent).

[0096] In some embodiments, the vaccine elicits a protective immune response in a human subject against diseases caused by Streptococcus pneumoniae infection.

[0097] According to a further aspect, the present disclosure provides a method for the prevention of Streptococcus pneumoniae infection or disease, the method comprising administering to a human subject a prophylactically effective amount of a mixed carrier 21-valent pneumococcal conjugate composition or a vaccine comprising the same. The mixed carrier 21-valent pneumococcal conjugate composition or a vaccine comprising the same can be administered by any route, including, for example, systemic or mucosal routes, as described in further detail below.

[0098] In certain embodiments, the human subject is an elderly subject, and the disease is pneumonia or invasive pneumococcal disease (IPD). In certain embodiments, the elderly subject is at least 50 years old. In other embodiments, the elderly subject is at least 55 years old. In yet other embodiments, the elderly subject is at least 60 years old.

[0099] In other embodiments, the human subject is an infant, and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM). In certain embodiments, the infant is between 0 and 2 years old. In other embodiments, the infant is between 2 and 15 months old.

[0100] In yet another embodiment, the human subject is between 6 weeks and 17 years old, and the disease is pneumonia, invasive pneumococcal disease (IPD) or acute otitis media (AOM). In certain embodiments, the human subject is between 6 weeks and 5 years old. In other embodiments, the human subject is between 5 and 17 years old.

[0101] The amount of the complex in each vaccine dose or the prophylactically effective amount of the mixed carrier multivalent pneumococcal conjugate composition can be selected as an amount that induces prophylaxis without significant side effects. Such amount can vary depending on the serotype of pneumococcus. Generally, each dose can contain from about 0.1 μg to about 100 μg, specifically from about 0.1 to 10 μg, more specifically from about 1 μg to about 5 μg of polysaccharide. The optimal amount of the components for a particular vaccine can be elucidated by standard studies including observation of an appropriate immune response in the subject. For example, the amount for vaccination of a human subject can be determined by extrapolating the results of animal tests. Also, the dose can be determined empirically.

[0102] In some embodiments, the vaccine or mixed carrier 21-valent pneumococcal conjugate composition is a single 0.5 ml dose formulated to contain from about 1 μg to about 5 μg of each capsular polysaccharide; from about 1 μg to about 30 μg of TT; from about 20 μg to about 85 μg of CRM 197 ; and optionally from about 0.1 mg to about 0.5 mg of elemental aluminum adjuvant. In some embodiments, the vaccine or mixed carrier 21-valent pneumococcal conjugate composition is a single 0.5 ml dose formulated to contain from about 4 μg to about 5 μg of serotype 6B and from about 2 μg to about 2.5 μg of each capsular polysaccharide excluding serotype 3 optionally; from about 2 μg to about 25 μg of TT; from about 40 μg to about 75 μg of CRM 197 ; and optionally from about 0.1 mg to about 0.25 mg of elemental aluminum adjuvant.

[0103] In some embodiments, the vaccine or mixed carrier 21-valent pneumococcal conjugate composition can be a single 0.5 ml dose formulated to contain about 2.2 μg of each capsular polysaccharide, except for serotype 6B which is present in an amount of about 4.4 μg.

[0104] In some embodiments, the vaccine or mixed carrier 21-valent pneumococcal conjugate composition can be a single 0.5 ml dose formulated to contain each of about 2 μg to about 2.5 μg of capsular polysaccharides, excluding up to 6 capsular polysaccharides selected from the group consisting of serotypes 1, 3, 4, 5, 6B, 9V, 19A, and 19F (each present in an amount of about 4 μg to about 5 μg). In one embodiment, the up to 6 capsular polysaccharides present in an amount of about 4 μg to about 5 μg are selected from the group consisting of serotypes 1, 3, 4, 6B, 9V, 19A, and 19F. In other embodiments, the vaccine or mixed carrier 21-valent pneumococcal conjugate composition can be a single 0.5 ml dose formulated to contain each of about 2.2 μg of capsular polysaccharides, excluding up to 6 capsular polysaccharides selected from the group consisting of serotypes 1, 3, 4, 5, 6B, 9V, 19A, and 19F (each present in an amount of about 4.4 μg). In one embodiment, the up to 6 capsular polysaccharides present in an amount of about 4.4 μg are selected from the group consisting of serotypes 1, 3, 4, 6B, 9V, 19A, and 19F.

[0105] In some embodiments, the vaccine or mixed carrier 21-valent pneumococcal conjugate composition can be a single 0.5 ml dose formulated to contain about 2 μg to about 2.5 μg of capsular polysaccharides of serotypes 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 22F, 23F, and 33F and about 4 μg to about 5 μg of capsular polysaccharides of serotypes 1, 3, 6B, 19A, and 19F.

[0106] In some embodiments, the vaccine or mixed carrier 21-valent pneumococcal conjugate composition may be a single 0.5 ml dose formulated to contain about 2 μg to about 2.5 μg of capsular polysaccharides of serotypes 1, 5, 6A, 7F, 8, 9N, 10A, 11A, 12F, 14, 15B, 18C, 22F, 23F, and 33F and about 4 μg to about 5 μg of capsular polysaccharides of serotypes 3, 4, 6B, 9V, 19A, and 19F.

[0107] In certain embodiments, the vaccine or mixed carrier 21-valent pneumococcal conjugate composition may be a single 0.5 ml dose formulated to contain about 2 - 2.5 μg of capsular polysaccharides of serotypes 1, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F and about 4 - about 5 μg of capsular polysaccharides of serotypes 3 and 6B.

[0108] In some embodiments, the vaccine or mixed carrier 21-valent pneumococcal conjugate composition may be a single 0.5 ml dose formulated to contain about 2 - about 2.5 μg of capsular polysaccharides of serotypes 1, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F and about 4 - about 5 μg of capsular polysaccharides of serotype 6B and about 8 - about 9 μg of capsular polysaccharides of serotype 3, more preferably about 8.8 μg of capsular polysaccharides of serotype 3.

[0109] In certain embodiments, the mixed carrier 21-valent pneumococcal conjugate composition or a vaccine containing the same further comprises sodium chloride and sodium succinate buffer as excipients.

[0110] In some embodiments, in the mixed carrier 21-valent pneumococcal conjugate composition, each of the pneumococcal capsular polysaccharides of serotypes 1, 5, 15B, and 22F is conjugated to TT, and the capsular polysaccharides derived from serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, and 33F are CRM 197It can be formulated into a (21-valent) liquid formulation complexed with. Each 0.5 mL dose contains approximately 2.2 μg of each capsular polysaccharide except for approximately 4.4 μg of serotype 6B, approximately 2 μg to approximately 25 μg of TT carrier protein (only for serotypes 1, 5, 15B, and 22F), and approximately 40 μg to approximately 75 μg of CRM 197 carrier protein, approximately 0.125 to 0.250 mg of elemental aluminum (approximately 0.5 to 1.2 mg of aluminum phosphate) adjuvant, and a liquid containing sodium chloride and sodium succinate buffer as excipients. It can be formulated into a liquid.

[0111] In some embodiments, in the mixed carrier 21-valent pneumococcal conjugate composition, each of the pneumococcal capsular polysaccharides of serotypes 3, 5, 15B, and 22F is complexed to TT, and the capsular polysaccharides derived from serotypes 1, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, and 33F are complexed to CRM 197 It can be formulated into a (21-valent) liquid formulation complexed with. In one embodiment, each 0.5 mL dose contains approximately 2.2 μg of each capsular polysaccharide except for approximately 4.4 μg of serotype 6B, approximately 2 μg to approximately 25 μg of TT carrier protein (only for serotypes 3, 5, 15B, and 22F), and approximately 40 μg to approximately 70 μg of CRM 197 carrier protein, approximately 0.125 mg to approximately 0.250 mg of elemental aluminum (approximately 0.5 to 1.2 mg of aluminum phosphate) adjuvant, and a liquid containing sodium chloride and sodium succinate buffer as excipients. In another embodiment, each 0.5 mL dose contains approximately 2.2 μg of each capsular polysaccharide excluding up to 6 capsular polysaccharides selected from the group consisting of serotypes 1, 3, 4, 5, 6B, 9V, 19A, and 19F except, approximately 2 μg to approximately 25 μg of TT carrier protein (only for serotypes 3, 5, 15B, and 22F), and approximately 40 μg to approximately 70 μg of CRM 197It can be formulated into a liquid containing a carrier protein, about 0.125 mg to about 0.250 mg of elemental aluminum (about 0.5 to 1.2 mg of aluminum phosphate) adjuvant, and sodium chloride and sodium succinate buffer as excipients. In one embodiment, up to 6 capsular polysaccharides of about 4.4 μg are selected from the group consisting of serotypes 1, 3, 4, 6B, 9V, 19A, and 19F. In yet another embodiment, each 0.5 mL dose contains about 2.2 μg of each capsular polysaccharide except for serotypes 1, 3, 6B, 19A, and 19F at about 4.4 μg, about 2 μg to about 25 μg of TT carrier protein (only for serotypes 3, 5, 15B, and 22F), and about 40 μg to about 70 μg of CRM 197 It can be formulated into a liquid containing a carrier protein, about 0.125 mg to about 0.250 mg of elemental aluminum (about 0.5 to 1.2 mg of aluminum phosphate) adjuvant, and sodium chloride and sodium succinate buffer as excipients.

[0112] In some embodiments, in the mixed carrier 21-valent pneumococcal conjugate composition, each of the pneumococcal capsular polysaccharides of serotypes 1, 3, 15B, and 22F is conjugated to TT, and the capsular polysaccharides from serotypes 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, and 33F are conjugated to CRM 197 It can be formulated into a (21-valent) liquid formulation in which they are conjugated to CRM. In one embodiment, each 0.5 mL dose contains about 2.2 μg of each capsular polysaccharide except for serotype 6B at about 4.4 μg, about 2 μg to about 25 μg of TT carrier protein (only for serotypes 1, 3, 15B, and 22F), and about 40 μg to about 75 μg of CRM 197 It can be formulated into a liquid containing a carrier protein, about 0.125 mg to about 0.250 mg of elemental aluminum (about 0.5 to 1.2 mg of aluminum phosphate) adjuvant, and sodium chloride and sodium succinate buffer as excipients.

[0113] In some embodiments, the liquid formulation can be filled into a single-dose syringe without using a preservative. After shaking, the liquid formulation becomes a vaccine that is a homogeneous white suspension prepared for intramuscular administration.

[0114] The mixed carrier 21-valent pneumococcal conjugate composition can be administered as a single injection or as part of an immunization series. For example, the mixed carrier 21-valent pneumococcal conjugate composition can be administered 2, 3, 4, or more times at appropriate intervals, such as intervals of 1, 2, 3, 4, 5, or 6 months or combinations thereof. In some embodiments, the mixed carrier 21-valent pneumococcal conjugate composition is administered to infants 4 times within the first 15 months of life, including, for example, at about 2, 3, 4, and 12 - 15 months of age, at about 3, 4, 5, and 12 - 15 months of age, or at about 2, 4, 6, and 12 - 15 months of age. This first dose can be administered as early as 6 weeks of age. In another embodiment, the mixed carrier 21-valent pneumococcal conjugate composition is administered to infants 3 times within the first 15 months of life, including, for example, at about 2, 4, and 11 - 12 months of age.

[0115] The mixed carrier multivalent pneumococcal conjugate composition can also include one or more proteins derived from Streptococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for inclusion include those identified in International Patent Application WO02 / 083855 and those described in International Patent Application WO02 / 053761.

[0116] The mixed carrier 21-valent pneumococcal conjugate composition is administered to a subject via one or more administration routes known to those skilled in the art, such as parenteral, transdermal, or transmucosal, nasal, intramuscular, intraperitoneal, intradermal, intravenous, or subcutaneous routes, and can be formulated accordingly. The mixed carrier 21-valent pneu mococcal conjugate composition can be formulated to be compatible with its intended administration route.

[0117] In some embodiments, the mixed carrier 21-valent pneumococcal conjugate composition can be administered as a liquid formulation by intramuscular, intraperitoneal, subcutaneous, intravenous, intraarterial, or transdermal injection or respiratory mucosal injection. The mixed carrier 21-valent pneumococcal conjugate composition can be formulated in a liquid form or a lyophilized form. In some embodiments, the injectable composition is prepared in a conventional form as a liquid solution or suspension, as a solid form suitable for solution or suspension in a liquid prior to injection, or as an emulsion. In some embodiments, the injection solutions and suspensions are prepared from sterile powders or granules. General considerations in the formulation and manufacture of pharmaceutical agents for administration by these routes are described, for example, in Remington’s Pharmaceutical Sciences, 19 thSee, e.g., Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA, 1995 (incorporated herein by reference). Currently, oral or intranasal sprays or aerosol routes (e.g., by inhalation) are most commonly used to deliver therapeutic agents directly to the lungs and respiratory system. In some embodiments, the mixed carrier 21-valent pneumococcal conjugate composition is administered using a device that delivers a measured dose of the composition. Suitable devices for use in delivering the intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Patent No. 4,886,499, U.S. Patent No. 5,190,521, U.S. Patent No. 5,328,483, U.S. Patent No. 5,527,288, U.S. Patent No. 4,270,537, U.S. Patent No. 5,015,235, U.S. Patent No. 5,141,496, U.S. Patent No. 5,417,662 (all of which are incorporated herein by reference). The intradermal composition can also be administered by a device that limits the effective penetration length of the needle into the skin, such as those described in WO1999 / 34850, incorporated herein by reference, and functional equivalents thereof. Also suitable are jet injection devices that deliver a liquid vaccine to the dermis via a liquid jet syringe or via a needle that generates a jet that penetrates the stratum corneum and reaches the dermis. Jet injection devices are described, for example, in U.S. Patent No. 5,480,381, U.S. Patent No. 5,599,302, U.S. Patent No. 5,334,144, U.S. Patent No. 5,993,412, U.S. Patent No. 5,649,912, U.S. Patent No. 5,569,189, U.S. Patent No. 5,704,911, U.S. Patent No. 5,383,851, U.S. Patent No. 5,893,397, U.S. Patent No. 5,466,220, U.S. Patent No. 5,339,163, U.S. Patent No. 5,312,335, U.S. Patent No. 5,503,627, U.S. Patent No. 5,064,413, U.S. Patent No. 5,520,639, U.S. Patent No. 4,596,556, U.S. Patent No. 4,790,824, U.S. Patent No. 4,941,880, U.S. Patent No. 4,940,460, WO1997 / 37705, and WO1997 / 13537 (all of which are incorporated herein by reference).Also suitable is a ballistic powder / particle delivery device that uses compressed gas to accelerate vaccine in powder form through the outer layer of the skin to the dermis. Also, conventional syringes can be used in the classical Mantoux method of intradermal administration.

[0118] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, oils such as olive oil, and organic esters for injection such as ethyl oleate. Examples of oils include vegetable or animal oils, peanut oil, soybean oil, olive oil, sunflower oil, cod liver oil, synthetic oils such as marine oils, and lipids obtained from milk or eggs. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions (including physiological saline and buffer media). Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluids and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose). For example, preservatives and other additives such as antibacterial agents, antioxidants, chelating agents, and inert gases may also be present.

[0119] The 21-valent pneumococcal conjugate composition can be formulated in the form of a unit-dose vial, a multi-dose vial, or a pre-filled syringe. Pharmaceutically acceptable carriers for liquid formulations include aqueous or non-aqueous solvents, suspensions, emulsions, or oils. The composition can be isotonic, hypertonic, or hypotonic. However, it is preferably basically isotonic for infusion or injection compositions. Therefore, isotonicity or hypertonicity can be advantageous for the storage of the composition. If the composition is hypertonic, it can be diluted to isotonicity before administration. The isotonicity agent can be an ionic isotonicity agent such as a salt or a non-ionic isotonicity agent such as a carbohydrate. Ionic isotonicity agents include, but are not limited to, sodium chloride, calcium chloride, potassium chloride, and magnesium chloride. Non-ionic isotonicity agents include, but are not limited to, sorbitol and glycerol. Preferably, at least one pharmaceutically acceptable buffer is included. For example, when the composition is an infusion or injection, it is preferably formulated in a buffer having a buffering capacity at pH 4 to pH 10, for example, pH 5 to pH 9, or pH 6 to pH 8. The buffer can be selected from those suitable for the United States Pharmacopeia (USP). For example, the buffer can be selected from the group consisting of monobasic acids such as acetic acid, benzoic acid, gluconic acid, glyceric acid, and lactic acid; dibasic acids such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, and tartaric acid; polybasic acids such as citric acid and phosphoric acid; and bases such as ammonia, diethanolamine, glycine, triethanolamine, and TRIS.

[0120] The 21-valent pneumococcal conjugate composition may contain a surfactant. Examples of surfactants include polyoxyethylene sorbitan esters (commonly referred to as Tween), in particular, polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and butylene oxide (BO) (such as DOWFAX); octoxynol having different repeats of ethoxy (oxy-1,2-ethanediyl) groups, in particular, octoxynol-9 (Triton-100); ethylphenoxypolyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as lecithin; nonylphenol ethoxylates such as the TERGITOL NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl, oleyl alcohol (Brij surfactants), in particular, triethylene glycol monolauryl ether (Brij30); sorbitan ethers known as SPAN® , in particular, sorbitan trioleate (Span85) and sorbitan monolaurate, but are not limited thereto.

[0121] Mixtures of surfactants such as Tween80 / Span85 can be used. Combinations of polyoxyethylene sorbitan esters such as Tween80 and octoxynol such as Triton X-100 are also suitable. Combinations of Laureth9 with Tween and / or octoxynol are also advantageous. Preferably, the amount of polyoxyethylene sorbitan ester (such as Tween80) contained can be 0.01% - 1% (w / v), 0.01% - 0.1% (w / v), 0.01% - 0.05% (w / v), or about 0.02%; the amount of octylphenoxypolyoxyethanol or nonylphenoxypolyoxyethanol (such as Triton X-100) contained can be 0.001% - 0.1% (w / v), particularly 0.005% - 0.02%; the amount of polyoxyethylene ether (such as Laureth9) contained can be 0.1% - 20% (w / v), optionally 0.1% - 10%, particularly 0.1% - 1% or about 0.5%.

[0122] In some embodiments, the mixed carrier 23-valent pneumococcal conjugate composition can be delivered via a controlled release system. For example, intravenous infusion, transdermal patch, liposome, or other routes can be used for administration. In one aspect, macromolecules such as microspheres or implants can be used.

[0123] The above disclosure generally describes the present invention. A more complete understanding can be obtained by referring to the following specific examples. These examples are described for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples

[0124] Example 1. Preparation of S. pneumoniae capsular polysaccharide

[0125] The culture of S. pneumoniae and the purification of the capsular polysaccharide were performed as known to those skilled in the art. S. pneumoniae serotypes were obtained from the American Type Culture Collection (ATCC) (serotype 1: ATCC number 6301; serotype 3: ATCC number 6303; serotype 4: ATCC number 6304; serotype 5: ATCC number 6305; serotype 6A: ATCC number 6306; serotype 6B: ATCC number 6326; serotype 7F: ATCC number 10351; serotype 9N: ATCC number 6309; serotype 9V: ATCC number 10368; serotype 14: ATCC number 6314; serotype 18C: ATCC number 10356; serotype 19A: ATCC number 10357; serotype 19F: ATCC number 6319; serotype 23F: ATCC number 6323). In-house strains of serotypes 8, 10A, 11A, 12F, 15B, 22F, and 33F were used, but any publicly available strain could be used. S. pneumoniae was characterized by a capsule and motility, gram-positive, lanceolate diplococcus, and alpha-hemolysis on blood agar medium. Serotypes were identified by the Quelling test (U.S. Patent No. 5,847,112) using specific antisera.

[0126] Preparation of cell bank

[0127] The strain was propagated to generate several generations of seed stocks (generations F1, F2, and F3) to remove animal-derived components. Two additional generations of seed stocks were produced. The first additional generation was cultured from the F3 vial, and subsequent generations were cultured from vials of the first additional generation. The seed vials were cryopreserved using synthetic glycerol as a cryoprotectant (below -70 °C). For cell bank preparation, all cultures were grown in a soy-based medium. Before freezing, the cells were concentrated by centrifugation, the used medium was removed, and the cell pellet was resuspended in fresh medium containing a cryoprotectant (such as synthetic glycerol).

[0128] Cultivation and Harvest

[0129] Cultures from the working cell bank were seeded into seed bottles containing a soy-based medium and cultured. After reaching the target optical density (absorbance), the seed bottles were used to seed a fermenter containing a soy-based medium. The cultivation was terminated when the optical density value began to be maintained constantly. After the cultivation was terminated, sodium deoxycholate was added to the culture to lyse the cells. The contents of the fermenter obtained were cooled to induce precipitation of proteins. Then, the mixture was centrifuged to remove the precipitated proteins and cell debris.

[0130] Purification

[0131] The solution obtained from centrifugation was filtered through a depth filter to remove proteins and cell debris that did not precipitate during centrifugation. The filtrate was concentrated on a 100 kDa MW membrane, and the concentrate was diafiltered with 10 volumes of 25 mM sodium phosphate buffer (pH 7.2) to obtain a sample. The sample was filtered to collect the supernatant, from which polysaccharides were precipitated and filtered. The filtrate was concentrated on a 30 kDa membrane, and the concentrate was used with approximately 10 volumes of triple-distilled water Diafiltration was performed. After performing diafiltration, the remaining solution was filtered through a 0.2 μm filter. In-process control tests (appearance, residual protein, residual nucleic acid, endotoxin, molecular weight, and total amount of polysaccharide) were performed on the filtrate. The concentrate was aseptically filtered and stored at -20°C.

[0132] Example 2. Preparation of a complex of S. pneumoniae capsular polysaccharide and carrier protein

[0133] Polysaccharides of different serotypes were activated according to different routes and then complexed with the carrier proteins CRM 197 or TT. Specifically, complexes were prepared by complexing each of the capsular polysaccharides of all serotypes except 15B and 22F with CRM 197 and by complexing each of the capsular polysaccharides of serotypes 1, 3, 5, 15B, and 22F with TT. Depending on the size of the native serotype, the activation process may include a decrease in the size of each capsular polysaccharide to the target molecular weight, chemical activation, and buffer exchange via ultrafiltration. The complexes were purified using ultrafiltration and finally filtered through a 0.2 μm filter. The process parameters such as pH, temperature, concentration, and time were as follows.

[0134] (1) Activation process

[0135] Step 1: Hydrolysis

[0136] Reductive amination is a known method for complexing polymers in which an amide bond is formed between the primary amine (-NH 2 ) group of a protein and the aldehyde of a saccharide. An aldehyde group is added to the pneumococcal capsular polysaccharide to facilitate complexation with the carrier protein. The vicinal diol structure of monosaccharides can be oxidized by sodium periodate (NaIO 4 ) to form an aldehyde group. The capsular polysaccharides of serotypes 1, 3, 4, 6A, 8, 11A, 12F, 14, 15B, 18C, 22F, and 33F were pretreated as follows.

[0137] For serotype 1, sodium hydroxide (final base concentration of 0.05 M) was added to the solution of capsular polysaccharide, and the solution was incubated at 50 ± 2 °C. Then the solution was cooled to a temperature in the range of about 21 °C to about 25 °C, and hydrochloric acid was added thereto so that the final pH became 6.0 ± 0.1, thereby stopping the hydrolysis.

[0138] For serotypes 3, 8, 11A, and 15B, hydrochloric acid (final acid concentration of 0.01 M) was added to the solution of capsular polysaccharide, and the solution was incubated at 60 ± 2 °C. Then the solution was cooled to a temperature in the range of about 21 °C to about 25 °C, and 0.1 M sodium phosphate was added thereto so that the final pH became 6.0 ± 0.1, thereby stopping the hydrolysis.

[0139] For serotype 4, hydrochloric acid (final acid concentration of 0.1 M) was added to the solution of capsular polysaccharide, and the solution was incubated at 45 ± 2 °C. Then the solution was cooled to a temperature in the range of about 21 °C to about 25 °C, and 1 M sodium phosphate was added thereto so that the final pH became 6.0 ± 0.1, thereby stopping the hydrolysis.

[0140] For serotype 6A, glacial acetic acid (final acid concentration of 0.1 M) was added to the solution of capsular polysaccharide, and the solution was incubated at 60 ± 2 °C. Then the solution was cooled to a temperature in the range of about 21 °C to about 25 °C, and 1 M sodium hydroxide was added thereto so that the final pH became 6.0 ± 0.1, thereby stopping the hydrolysis.

[0141] For serotype 12F, hydrochloric acid (final acid concentration of 0.01 M) was added to the solution of capsular polysaccharide, and the solution was incubated at 70 ± 2 °C. Then the solution was cooled to a temperature in the range of about 21 °C to about 25 °C, and 0.1 M sodium phosphate was added thereto so that the final pH became a solution of 6.0 ± 0.1 as described above, thereby stopping the hydrolysis.

[0142] For serotypes 14 and 18C, glacial acetic acid (final acid concentration of 0.2 M) was added to the solution of capsular polysaccharide, and the solution was incubated at 94 ± 2 °C. The solution was then cooled to a temperature in the range of about 21 °C to about 25 °C, and 1 M sodium phosphate was added thereto to a final pH of the solution of 6.0 ± 0.1, thereby stopping the hydrolysis.

[0143] For serotypes 22F and 33F, hydrochloric acid (final acid concentration of 0.01 M) was added to the solution of capsular polysaccharide, and the solution was incubated at 60 ± 2 °C. The solution was then cooled to a temperature in the range of about 21 °C to about 25 °C, and 0.1 M sodium phosphate was added thereto to a final pH of 6.0 ± 0.1, thereby stopping the hydrolysis.

[0144] Each of the obtained capsular polysaccharides was diluted in water for injection (WFI), sodium acetate, and sodium phosphate to a final concentration of about 1.0 mg / mL to about 2.0 mg / mL.

[0145] Step 2: Periodate reaction

[0146] The molar equivalent of sodium periodate for the activation of each pneumococcal polysaccharide was determined based on the molar mass of the repeating unit. Oxidation reactions were allowed to proceed at 21 °C to 25 °C for 16 to 20 hours for all serotypes except 1, 7F, and 19F, which were well mixed and brought to a temperature of 10 °C or less. To assist in maintaining consistent and stable production of the conjugate, the range of degree of oxidation (Do) levels for each serotype is targeted during the conjugation process. The preferred target ranges of Do levels for each serotype are shown in Tables 1 and 2. [Table 1] [Table 2]

[0147] Step 3: Ultrafiltration

[0148] The acidified saccharide was concentrated, and diafiltration was performed using WFI with a 100 kDa MWCO ultrafiltration membrane (a 30 kDa ultrafiltration membrane for serotype 1 and a 5 kDa ultrafiltration membrane for serotype 18C). For serotype 1, diafiltration was carried out using a 0.9% sodium chloride solution; for serotypes 7F and 23F, a 0.01 M sodium acetate buffer (pH 4.5); and for serotype 19F, a 0.01 M sodium phosphate buffer (pH 6.0). The permeate was discarded, and the retentate was filtered through a 0.2 μm filter.

[0149] Step 4: Lyophilization

[0150] In the case of the capsular polysaccharides of serotypes 3, 4, 5, 8, 9N, 9V, 10A, 14, and 33F that are complexed with carrier proteins by using an aqueous solvent, a mixed solution of the polysaccharide and the carrier protein was prepared without adding additional sucrose, lyophilized, and then stored at -25°C ± 5°C.

[0151] In the case of the capsular polysaccharides of serotypes 1 and 18C that are complexed with carrier proteins by using an aqueous solvent, the polysaccharide and the carrier protein were prepared independently without adding additional sucrose, lyophilized, and then stored at -25°C ± 5°C.

[0152] In the case of the capsular polysaccharides of serotypes 6A, 6B, 7F, 15B-TT, 19A, 19F, 22F-TT, and 23F that are complexed with carrier proteins by using a DMSO solvent, a predetermined amount of sucrose to reach a final sucrose concentration of 5% ± 3% (w / v) was added to the activated saccharide, the samples were prepared independently, lyophilized, and then stored at -25°C ± 5°C.

[0153] In the case of the capsular polysaccharide of serotype 11A, a predetermined amount of sucrose to reach a final sucrose concentration of 20% ± 5% (w / v) was added to the activated saccharide, the polysaccharide and the carrier protein were prepared independently, lyophilized, and then stored at -25°C ± 5°C.

[0154] For the capsular polysaccharide of serotype 12F, a predetermined amount of sucrose to reach a final sucrose concentration of 10% ± 5% (w / v) was added to the activated polysaccharide, and the polysaccharide and carrier protein were prepared independently, lyophilized, and then stored at -25°C ± 5°C.

[0155] (2) Complexation process

[0156] For serotypes 1, 3, 4, 5, 8, 9N, 9V, 10A, 14, 18C, and 33F, aqueous complexation was performed, and for serotypes 6A, 6B, 7F, 11A, 12F, 15B-TT, 19A, 19F, 22F-TT, and 23F, DMSO complexation was performed. Each of the capsular polysaccharides was complexed with the carrier protein at a ratio of 0.2 - 2:1.

[0157] Step 1: Dissolution

[0158] Aqueous complexation

[0159] For serotypes 1, 3, 4, 5, 8, 9N, 9V, 10A, 14, 18C, and 33F, the lyophilized samples were thawed and equilibrated at room temperature. The lyophilized samples were reconstituted in the reaction concentrate at 23 ± 2°C at the ratio set for each serotype by using sodium phosphate buffer solution.

[0160] Dimethyl sulfoxide (DMSO) complexation

[0161] For serotypes 6A, 6B, 7F, 11A, 12F, 15B-TT, 19A, 19F, 22F-TT, and 23F, the lyophilized samples were thawed, equilibrated at room temperature, and reconstituted in DMSO.

[0162] Step 2: Complexation reaction

[0163] Aqueous complexation

[0164] For serotypes 3-TT, 4, 5-TT, 8, 9N, 9V, 10A, 14, 18C, and 33F, the complexation reaction was initiated by adding sodium cyanoborohydride solution (100 mg / mL) to give 1.0 to 1.4 moles of sodium cyanoborohydride per mole of saccharide. However, for serotypes 1, 1-TT, and 3, the reaction was initiated by adding sodium cyanoborohydride solution to give 0.5 moles of sodium cyanoborohydride per mole of saccharide.

[0165] The reaction mixture was incubated at 23 °C to 37 °C for 44 to 106 hours. The reaction temperature and time were adjusted according to the serotype. The temperature was then lowered to 23 ± 2 °C, and 0.9% sodium chloride was added to the reactor. Sodium borohydride solution (100 mg / mL) was added to give 1.8 to 2.2 molar equivalents of sodium borohydride per mole of saccharide. The mixture was incubated at 23 ± 2 °C for 3 to 6 hours. This procedure reduced the unreacted aldehyde present on the saccharide. The mixture was then diluted with 0.9% sodium chloride, and the diluted complexation mixture was filtered using a 0.8 or 0.45 μm prefilter.

[0166] DMSO Complexation

[0167] For the capsular polysaccharides of serotypes 6A, 6B, 7F, 11A, 12F, 15B-TT, 19A, 19F, 22F-TT, and 23F, the complexation reaction was initiated by adding sodium cyanoborohydride solution (100 mg / mL) to give 0.8 to 1.2 molar equivalents of sodium cyanoborohydride per mole of activated saccharide. WFI was added to the reaction mixture to a target concentration of 1% (v / v), and the mixture was incubated at 23 ± 2 °C for 12 to 26 hours. 100 mg / mL sodium borohydride solution (typically 1.8 to 2.2 molar equivalents of sodium borohydride per mole of activated saccharide) and WFI (target 5% v / v) was added to the reactants, and the mixture was incubated at 23 ± 2 °C for 3 - 6 hours. This procedure reduced the unreacted aldehydes present on the glycoprotein. The reaction mixture was then diluted with 0.9% sodium chloride, and the diluted complexation mixture was filtered using a 0.8 or 0.45 μm prefilter.

[0168] Step 3: Ultrafiltration

[0169] The diluted complex mixture was concentrated, and diafiltration was performed using at least 15 volumes of 0.9% sodium chloride or buffer with a 100 kDa MWCO ultrafiltration filter or a 300 kDa MWCO ultrafiltration filter. Also, the composition and pH of the buffer used in the process varied depending on each serotype.

[0170] Step 4: Sterile Filtration

[0171] The retentate after ultrafiltration was sterile filtered (0.2 μm), and in - process control (appearance, free protein, free glycoprotein, molecular size distribution, sterility, glycoprotein content, protein content, pH, endotoxin, residual cyanide, residual DMSO, glycoprotein identity, TT identity, and CRM 197 identity) was performed on the filtered complex. The final concentrate was refrigerated and stored at 2 °C - 8 °C.

[0172] Example 3. Formulation of the polyvalent pneumococcal conjugate vaccine

[0173] The desired volume of the final bulk concentrate obtained from Example 2 was calculated based on the batch volume and the bulk saccharide concentration. After adding 0.85% sodium chloride (physiological saline), polysorbate 80, and succinate buffer to a pre-labeled formulation container, the bulk concentrate was added. The preparation was then mixed well and sterile filtered through a 0.2 μm membrane. The formulated bulk was gently mixed during and after the addition of aluminum hydroxide phosphate. The pH was checked and adjusted as necessary. The formulated bulk product was stored at 2 - 8°C. The following non-limiting multivalent pneumococcal conjugate vaccine formulations were prepared and named PCV21(1 / 5 / 15B / 22F)-TT and PCV21(3 / 5 / 15B / 22F)-TT.

[0174] PCV21(1 / 5 / 15B / 22F)-TT was prepared by conjugating each polysaccharide of serotypes 1, 5, 15B, and 22F to TT, and also conjugating each polysaccharide of serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, and 33F to CRM 197 and contained a polysaccharide-conjugate prepared by conjugation to CRM.

[0175] PCV21(3 / 5 / 15B / 22F)-TT was prepared by conjugating each polysaccharide of serotypes 3, 5, 15B, and 22F to TT, and also conjugating each polysaccharide of serotypes 1, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, and 33F to CRM 197 and contained a polysaccharide-conjugate prepared by conjugation to CRM.

[0176] The 0.5 ml total dose of the PCV21(1 / 5 / 15B / 22F)-TT composition contained 2.2 μg of each polysaccharide except 4.4 μg of serotype 6B, 2 μg - 25 μg of TT (for serotypes 1, 5, 15B, and 22F), and 40 μg - 75 μg of CRM 197, 0.125 mg of elemental aluminum (0.5 mg of aluminum phosphate) adjuvant, 4.25 mg of sodium chloride, approximately 295 μg of succinate buffer; and approximately 100 μg of polysorbate 80 were included in a total dose of 0.5 ml.

[0177] The 0.5 ml total dose of PCV21(3 / 5 / 15B / 22F)-TT composition contained 2.2 μg of each polysaccharide except for 4.4 μg of serotypes 1, 3, 6B, 19A, and 19F, 2 μg - 25 μg of TT (for serotypes 3, 5, 15B, and 22F), 0.250 mg of elemental aluminum (1.13 mg of aluminum phosphate), and had the same other components and their contents as that of PCV21(1 / 5 / 15B / 22F)-TT.

[0178] Example 4. Immunogenicity of the polyvalent pneumococcal conjugate vaccine The ability of PCV21(1 / 5 / 15B / 22F)-TT and PCV21(3 / 5 / 15B / 22F)-TT, which are the mixed carrier polyvalent pneumococcal vaccines prepared in Example 3, to induce an immunogenic response in rabbits was tested. The evaluation of immunogenicity was carried out by antigen-specific ELISA for serum IgG concentration and by opsonophagocytosis assay (OPA) for antibody functionality. New Zealand white rabbits were intramuscularly immunized at week 0 and week 2 with each polysaccharide at a dose 5% higher than the planned human clinical dose in the formulation (2.31 μg of each polysaccharide except 4.62 μg of 6B) or the human dose (2.2 μg of each polysaccharide except 4.4 μg of 6B). Serum was collected every 2 weeks after immunization. The same results were shown at any concentration.

[0179] 4-1. PCV21(3 / 5 / 15B / 22F)-TT

[0180] Determination of serotype-specific IgG concentration

[0181] The capsular polysaccharides (PnP) for each serotype were coated on a 96-well plate at 0.5 μg / well to 1 μg / well. An equivalent amount of serum was collected from each subject and pooled by group. The serum pool was serially diluted 2.5-fold with an antibody dilution buffer containing Tween20 and pneumococcal cell wall polysaccharide (CWPS) (5 μg / mL) obtained from Statens Serum Institut, and then reacted at room temperature for 30 minutes. The plate was washed 5 times with a wash buffer, and then 50 μl of pre-adsorbed and diluted serum was added to the coated well plate, followed by incubation at room temperature for 2 to 18 hours. After washing the well plate in the same manner, then a goat anti-rabbit IgG-alkaline phosphatase conjugate was added to each well, followed by incubation at room temperature for 2 hours. The plate was washed as described above, and 1 mg / mL p-nitrophenylamine buffer was added to each well as a substrate and reacted at room temperature for 2 hours. The reaction was quenched by adding 50 μl of 3M NaOH, and the absorbance at 405 nm and 690 nm was measured. As a comparative example, a commercially available 13-valent vaccine (PREVNAR13) was subjected to the same procedure. The results are shown in Table 3.

[0182]

Table 3

[0183] When the capsular polysaccharides of serotypes 3 and 5 were conjugated to TT, the serotype-specific IgG concentration was significantly increased compared to that obtained when conjugated to CRM 197 . Also, rabbits immunized with PCV21 (3 / 5 / 15B / 22F)-TT also demonstrated a significant increase in IgG concentration against 8 additional serotypes not present in PREVNAR13 (i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F). In particular, serotype 9N had an increase in serotype-specific IgG concentration exceeding 50-fold compared to PREVNAR13.

[0184] Functional immunogenicity test (MOPA)

[0185] Antibody function was evaluated by testing sera in the MOPA assay. S. pneumoniae MOPA strains stored at -70°C or below were diluted to the corresponding final dilution factor such that the concentration of each strain was approximately 50,000 CFU / mL. Equivalent amounts of sera were collected from each subject, pooled by group, and serially diluted 2-fold so that 20 μl of serum remained in the U-bottom plate. After diluting the samples, 10 μl of the strain prepared for each serotype was mixed with the diluted samples, and the mixture was reacted at room temperature for 30 minutes so that S. pneumoniae and the antibody were well mixed. A mixture of pre-differentiated HL-60 cells and complement was added and reacted in an incubator (37°C) for 45 minutes. The temperature was lowered to stop phagocytosis, and 10 μl of the reaction solution was spotted onto an agar plate that had been pre-dried for 30 - 60 minutes, and then absorbed onto the plate for 20 minutes until dry. A 25 mg / mL TTC stock solution was added to the prepared overlay agar, and the appropriate antibody for the corresponding strain was added thereto. The mixture was thoroughly mixed, and then approximately 25 mL of the mixture was added to the plate and allowed to harden for about 30 minutes. The completely hardened plate was incubated in an incubator (37°C) for 12 - 18 hours, and then the colonies were counted. The MOPA titer was expressed as the dilution rate at which 50% killing was observed. As a comparative example, a commercially available 13-valent vaccine (PREVNAR13) was subjected to the same procedure. The results are shown in Table 4. 2 When serotypes 3 and 5 were conjugated to TT, the functional MOPA titer was significantly increased compared to the MOPA titer obtained when conjugated to CRM. Also, PCV21 ( 2 When serotypes 3 and 5 were conjugated to TT, the functional MOPA titer was significantly increased compared to the MOPA titer obtained when conjugated to CRM. Also, PCV21 (

[0186]

Table 4

[0187] When serotypes 3 and 5 were conjugated to TT, the functional MOPA titer was significantly increased compared to the MOPA titer obtained when conjugated to CRM. Also, PCV21( 197 When serotypes 3 and 5 were conjugated to TT, the functional MOPA titer was significantly increased compared to the MOPA titer obtained when conjugated to CRM. Also, PCV21( Rabbits immunized with 3 / 5 / 15B / 22F)-TT also demonstrated a significant increase in functional MOPA titers against each of eight additional serotypes not present in PREVNAR13 (i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F).

[0188] 4-2. PCV21(1 / 5 / 15B / 22F)-TT

[0189] Serotype-specific IgG concentrations and functional immunogenic titers were measured by the same method as in 4-1, and the results of two separate experiments are shown below.

[0190] Measurement of serotype-specific IgG concentration

[0191]

Table 5

[0192] When the capsular polysaccharides of serotypes 1 and 5 were conjugated to TT, the serotype-specific IgG concentration increased significantly compared to that obtained when conjugated to CRM 197 In addition, rabbits immunized with PCV21(1 / 5 / 15B / 22F)-TT also demonstrated a significant increase in IgG concentrations against eight additional serotypes not present in PREVNAR13 (i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F). Again, serotype 9N showed a significant increase (more than 50-fold) against PRENAR13.

[0193] Functional immunogenicity test (MOPA)

[0194]

Table 6

[0195] When serotypes 1 and 5 were conjugated to TT, the functional MOPA titer was CRM 197It was significantly increased compared to the MOPA titer obtained when complexed. Also, rabbits immunized with PCV21(3 / 5 / 15B / 22F)-TT demonstrated a significant increase in the functional MOPA titer against each of eight additional serotypes not present in PREVNAR13, namely 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F.

[0196] Example 5. Additional details regarding the preparation of the ribosome - protein complex derived from Streptococcus pneumoniae serotype 9N

[0197] Preparation of the cell bank

[0198] Streptococcus pneumoniae serotype 9N (ATCC6309) was obtained from the American Type Culture Collection (ATCC). The seed stock was cultured for several generations for strain growth and removal of animal - derived components. The stock vials were kept in a freezer (< - 70 °C) with synthetic glycerol as a cryoprotectant. For the preparation of the cell bank, the cell culture was grown in a soybean - based medium. Before freezing, the cells were concentrated by centrifugation, the used medium was removed, and the cell pellet was resuspended in fresh medium containing a cryoprotectant (e.g., synthetic glycerol).

[0199] Fermentation

[0200] The culture from the cell bank was seeded into a seed bottle containing a soybean - based medium. The culture was incubated at a constant temperature without agitation until the growth conditions were met. The seed bottle was used to seed the culture into a seed fermenter containing a soybean - based medium while controlling the temperature, pH, and agitation speed. Fermentation was terminated after growth ceased or after reaching the operating capacity of the fermenter. After terminating fermentation by adding an inactivator, cell debris was removed using a combination of continuous - flow centrifugation and filtration.

[0201] Purification

[0202] The purification process of the pneumococcal polysaccharide consisted of multi-layer filtration, repeated concentration / diafiltration and filtration / elution.

[0203] Activation

[0204] The final polysaccharide concentration was adjusted to approximately 2.0 g / L by sequentially adding calculated amounts of WFI. The reaction pH was adjusted to approximately 6.0 as needed. After pH adjustment, the reaction temperature was adjusted to 21 - 25°C. To initiate oxidation, approximately 0.024 - 0.189 mg of sodium periodate per mg of sugar was added. The oxidation reaction was carried out at 21 - 25°C for 16 - 20 hours.

[0205] The activated polysaccharide was concentrated and diafiltered using a 100 kDa MWCO ultrafiltration membrane. Diafiltration was performed with WFI at 10 times the diafiltration volume. The purified activated polysaccharide was then stored at 2 - 8°C. The purified activated polysaccharide was characterized by (i) polysaccharide concentration determined by colorimetric assay, (ii) aldehyde concentration determined by colorimetric assay, (iii) degree of oxidation, and (iv) molecular weight measured by SEC-MALLS.

[0206] SEC-MALLS is used to determine the molecular weights of polysaccharides and polysaccharide-protein complexes. SEC is used to separate polysaccharides based on their hydrodynamic volume. A refractive index (RI) detector and a multi-angle laser light scattering (MALLS) detector are used to determine the molecular weight. When light reacts with a substance, the light is scattered. The amount of scattered light is related to the concentration, the square of the dn / dc (specific refractive index increment), and the molar mass of the material. The molecular weight is calculated based on the signal of the scattered light from the MALLS detector and the concentration signal from the RI detector.

[0207] The degree of oxidation (DO) of the activated polysaccharide is determined as the mole of the sugar repeating unit divided by the mole of the aldehyde. The mole of the sugar repeating unit is determined using various colorimetric techniques, such as the anthrone assay. And the mole of the aldehyde is determined by the Park-Johnson colorimetric analysis assay.

[0208] Using these above-mentioned techniques, it was determined that the activated Streptococcus pneumoniae serotype 9N capsular polysaccharide obtained by the above-mentioned method has a degree of oxidation of 2 to 19, more typically 5 to 10, and a molecular weight of about 200 to 700 kDa.

[0209] Complexation

[0210] The activated polysaccharide was mixed with the carrier protein CRM at a ratio of 0.5 to 2 g of CRM per 1 g of the activated polysaccharide. 197 Then, the mixed mixture was lyophilized. The lyophilized mixture of the activated polysaccharide and CRM 197 was stored at -20 °C. 197 The lyophilized mixture of the activated polysaccharide and CRM

[0211] The lyophilized mixture of the activated polysaccharide and CRM 197 was reconstituted in 0.1 M sodium phosphate solution and then mixed well. The final polysaccharide concentration in the reaction solution was about 10 to 20 g / L. After initiating the complexation by adding 1.0 to 1.2 molar equivalents of sodium cyanoborohydride (NaBH 3 CN) to the reaction mixture, the reaction was carried out at 35 to 39 °C for 44 to 52 hours. The complexation reaction was terminated by adding a 0.9% sodium chloride solution of the same volume as the complexation reaction solution and then adding 1.8 to 2.2 molar equivalents of sodium borohydride (NaBH 4 ) to cap the unreacted aldehyde. The capping reaction was carried out at 21 to 25 °C for 3 to 6 hours.

[0212] For concentration and diafiltration using a 100 kDa MWCO membrane, the complex solution was diluted with 0.9% sodium chloride solution. The diluted complex solution was filtered through a 0.8 - 0.45 μm filter and purified by concentration and diafiltration. Diafiltration using a 100 kDa MWCO membrane was performed using 0.9% sodium chloride solution at 15 - 40 times the diafiltration volume. After diafiltration was completed, the remaining solution was filtered through a 0.2 μm filter. The complex solution was diluted to a concentration of less than approximately 0.55 mg / mL, sterile filtered, and then stored at 2 - 8 °C.

[0213] The purified serotype 9N complex was characterized, in particular, by (i) the protein concentration determined by a colorimetric (Lowry) assay, (ii) the aldehyde concentration determined by a colorimetric assay, (iii) the polysaccharide to protein ratio, (iv) the molecular size distribution determined by size exclusion chromatography (CL - 4B), and (v) the molecular weight measured by SEC - MALLS.

[0214] Changes in the characteristics of the serotype 9N complex were observed while varying the degree of oxidation (DO). The results are summarized in Table 7.

[0215]

Table 7

[0216] Changes in the characteristics of the serotype 9N complex were observed while varying the mixing ratio of the activated polysaccharide and CRM 197 during lyophilization. The results are summarized in Table 8.

[0217]

Table 8

[0218] Changes in the characteristics of the serotype 9N complex were observed while varying the polysaccharide concentration in the complexation reaction solution. The results are summarized in Table 9.

[0219]

Table 9

[0220] Example 6. Analysis of Immunogenicity

[0221] CRM 197 was formulated into a monovalent conjugate composition containing a Streptococcus pneumoniae serotype 9N ribosome - protein conjugate complexed to CRM

[0222] The immunogenicity of the monovalent immunogenic compositions in Tables 11 - 13 was analyzed by ELISA. The serum concentration of serotype - specific IgG was determined

[0223] Five female New Zealand white rabbits weighing 2.5 - 3.5 kg were immunized intramuscularly at week 0 at the proposed human clinical dose (2.2 μg of conjugate; + 0.25 mg / mL of aluminum as AlPO 4 ). The rabbits were re - immunized at week 2 with the same dose of conjugate vaccine, and blood samples were collected at week 4. Serotype - specific ELISA was performed on the serum samples at week 0 and week 4

[0224] The analysis results are shown in Table 10. Rabbits immunized with the monovalent conjugate composition (conjugate number 8) showed a significant increase in the total IgG titer against serotype 9N. Rabbits immunized with other conjugates also showed a significant increase in the total IgG titer

[0225] Table 10 shows the results of measuring the IgG concentration after immunizing rabbits with conjugate number 8 in Table 8

[0226]

Table 10

[0227] Although one or more exemplary embodiments are described herein, various changes in form and detail may be made herein without departing from the spirit and scope of the inventive concept defined by the following claims, as will be understood by those skilled in the art. It is understood by those skilled in the art that various changes may be made herein. References

[0228] The following references are cited in this application and provide general information regarding the art and assays and other details discussed in this application. The following references are hereby incorporated by reference in their entirety into this specification.

[0229] [1] Prymula et al., The Lancet, 367:740 - 48 (2006).

[0230] [2] Vesikari et al., PIDJ, 28(4):S66 - 76 (2009).

[0231] [3] Dagan et al., Infection & Immunity, 5383 - 91 (2004).

[0232] [4] Juergens et al., Clinical and Vaccine Immunology, 21(9):1277 - 1281 (2014).

[0233] [5] Andrews et al., The Lancet, 14:839 - 846 (2014).

[0234] [6] Nurkka et al., Vaccine, 20:194 - 201 (2001).

[0235] [7] Levin and Stone, J. Immunol., 67:235 - 242 (1951).

[0236] [8]W.H.O. Manual for the Production and Control of Vaccines: Tetanus Toxoid, 1977 (BLG / UNDP / 77.2 Rev.I.)

[0237] [9]Didierlaurent et al., J. Immunol., 183:6186 - 6197 (2009).

Claims

1. An immunogenic serotype 9N conjugate comprising a protein carrier conjugated to a capsular polysaccharide derived from Streptococcus pneumoniae serotype 9N, wherein the protein carrier is CRM １９７ and the capsular polysaccharide derived from serotype 9N is conjugated to CRM in an activated state such that the capsular polysaccharide derived from serotype 9N has an oxidation degree of 2 to 19 or 5 to 10 and a molecular weight of 200 to 700 kDa １９７ -conjugated immunogenic serotype 9N conjugate.

2. The immunogenic serotype 9N complex according to claim 1, having a molecular weight of 500 to 4000 kDa.

3. The CRM of the capsular polysaccharide derived from serotype 9N in the immunogenic serotype 9N complex １９７ The immunogenic serotype 9N complex according to claim 1 or 2, wherein the ratio to is 0.1 to 5 (w / w).

4. The CRM of the capsular polysaccharide derived from serotype 9N in the immunogenic serotype 9N complex １９７ The immunogenic serotype 9N complex according to claim 3, wherein the ratio to is 0.5 to 2.5 (w / w).

5. 15 to 60% of the immunogenic serotype 9N complex has a K of 0.3 or less as measured on a CL-4B column ｄ The immunogenic serotype 9N complex according to claim 1, 2 or 4, having

6. The immunogenic serotype 9N complex according to claim 1, 2, or 4, wherein the degree of oxidation is 2 to 19.

7. The immunogenic serotype 9N complex according to claim 6, wherein the degree of oxidation is 5 to 10. **Claim 8**: An immunogenic serotype 9N complex according to claim 1, having a molecular weight of 500 to 4000 kDa, a molecular weight distribution of 15 to 60% as measured on a CL-4B column, and a saccharide / protein ratio of 0.5 to 2.5, with a K of 0.3 or less as measured on a CL-4B column. ｄ ​

9. A mixed carrier polyvalent pneumococcal conjugate composition comprising the immunogenic serotype 9N complex according to claim 1, 2, 4, 7, or 8.

10. The mixed carrier polyvalent pneumococcal conjugate composition according to claim 9, further comprising an adjuvant.

11. A method for preparing an immunogenic conjugate of Streptococcus pneumoniae serotype 9N, comprising: (a) lysing bacterial cells that produce the capsular polysaccharide of Streptococcus pneumoniae serotype 9N by fermenting it; (b) purifying the capsular polysaccharide of Streptococcus pneumoniae serotype 9N from the lysed bacterial cells; (c) activating the capsular polysaccharide of Streptococcus pneumoniae serotype 9N by reacting it with an oxidizing agent to achieve a degree of oxidation of 2 to 19 or 5 to 10; (d) forming an immunogenic serotype 9N complex by mixing the activated capsular polysaccharide of Streptococcus pneumoniae serotype 9N with CRM197. A method comprising the above steps.

12. CRM mixed in step (d) １９７ The method according to claim 11, wherein the reaction with a reducing agent forms a complex with the activated capsular polysaccharide of Streptococcus pneumoniae serotype 9N.

13. The method according to claim 11, wherein in step (c), 0.02 to 0.19 μg of periodate is reacted with 1 μg of the capsular polysaccharide of Streptococcus pneumoniae serotype 9N at 20 to 25 °C for 15 to 20 hours.

14. The method according to any one of claims 11 to 13, wherein the capsular polysaccharide of Streptococcus pneumoniae serotype 9N reacted with the oxidizing agent in step (c) has a molecular weight of 400 to 900 kDa.

15. In step (d), CRM １９７ The method according to any one of claims 11 to 13, wherein the activated Streptococcus pneumoniae serotype 9N capsular polysaccharide mixed with has a molecular weight of 200 to 700 kDa.

16. The method according to any one of claims 11 to 13, wherein the immunogenic serotype 9N complex formed in step (d) has a molecular weight of 500 to 4000 kDa.

17. CRM against the activated capsular polysaccharide of serotype 9N １９７ The method according to any one of claims 11 to 13, wherein the initial input ratio is 0.5 to 2.5:

1.

18. At least 15 to 60% of the immunogenic serotype 9N complex has a K of 0.3 or less as measured by a CL-4B column ｄ The method according to any one of claims 11 to 13, having

19. In step (c), 0.02 to 0.19 μg of periodate is reacted with 1 μg of the capsular polysaccharide of Streptococcus pneumoniae serotype 9N, and the immunogenic serotype 9N conjugate formed in step (d) has a molecular weight of 500 to 4000 kDa, a K of 0.3 or less measured on a CL-4B column ｄ with a molecular weight distribution of 15 to 60%, and a CRM １９７ / polysaccharide ratio of 0.5 to 2.5, the method according to any one of claims 11 to 13.

20. The method according to any one of claims 11 to 13, wherein the degree of oxidation is 5 to 10.

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