Multivalent pneumococcal polysaccharide-protein conjugate composition

Abstract

To provide compositions and methods for preventing Streptococcus pneumoniae infection or disease.SOLUTION: A mixed-carrier multivalent pneumococcal conjugate composition is provided, comprising a protein carrier conjugated to capsular polysaccharides derived from different serotypes of Streptococcus pneumoniae, the protein carrier being CRM197 or tetanus toxoid, four of the capsular polysaccharides being conjugated to tetanus toxoid, the remaining capsular polysaccharides being conjugated to CRM197, the four capsular polysaccharides conjugated to tetanus toxoid being two serotypes selected from the group consisting of serotypes 15B, 22F, and serotypes 1, 3, and 5.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and relies on the filing dates of U.S. Provisional Patent Application No. 62 / 626,509, filed February 5, 2018, and Korean Patent Application No. 10-2018-0045246, filed April 18, 2018, the entire disclosures of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION This application relates generally to mixed carrier multivalent pneumococcal conjugate compositions, vaccines comprising the same, and methods of using these compositions and vaccines for the prevention of Streptococcus pneumoniae infection or disease in a subject. [Background technology]

[0003] Streptococcus pneumoniae is a Gram-positive, lance-shaped, facultative anaerobic bacterium with over 90 known serotypes. Most S. pneumoniae serotypes have been shown to cause disease, with the 23 most common serotypes accounting for approximately 90% of invasive disease worldwide. Serotypes are classified based on the serological response to capsular polysaccharides, the most important virulence 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] Early pneumococcal vaccines contained combinations of capsular polysaccharides from different serotypes. While these vaccines could confer immunity against S. pneumoniae in patients with developed or healthy immune systems, they were ineffective in infants and young children, who lack developed immune systems, and in elderly subjects, who often have compromised immune function. To improve the immune response to pneumococcal vaccines, especially in infants and elderly subjects at higher risk of developing S. pneumoniae infection, capsular polysaccharides were conjugated to suitable carrier proteins to create pneumococcal conjugate vaccines. Conjugation to a suitable carrier protein converts capsular polysaccharides from T-cell-independent antigens to T-cell-dependent antigens. Thus, the immune response to the conjugated capsular polysaccharides involves T helper cells, which help induce a stronger and more rapid immune response upon re-exposure to the capsular polysaccharides.

[0005] There are at least two approaches to 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. Pfizer's PREVNAR series of vaccines uses CRM, a non-toxic variant of diphtheria toxoid with a single amino acid substitution of glycine to glutamic acid. 197 An example of a single-carrier approach is where different capsular polysaccharides are conjugated to a protein carrier. The 7-valent PREVNAR vaccine (PREVNAR) was first licensed in 2000 and contains capsular polysaccharides from the seven most prevalent serotypes: 4, 6B, 9V, 14, 18C, 19F, and 23F. A 13-valent vaccine, PREVNAR13, contains the CRM 197 The protein carrier now includes serotypes 1, 5, 7F, 3, 6A, and 19A. Protein carrier CRM is the single carrier used in the PREVNAR vaccine. 197 has never been used as part of a mixed carrier system in a pneumococcal conjugate vaccine.

[0006] A 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, with capsular polysaccharides from a particular serotype conjugated to one protein carrier and capsular polysaccharides from a different serotype conjugated to at least a second, different protein carrier. For example, GlaxoSmithKline has developed SYNFLORIX, a 10-valent (serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F) mixed-carrier pneumococcal conjugate vaccine that uses H 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 demonstrate serotype-specific efficacy in acute otitis media trials. [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 and tetanus toxoids as protein carriers. [3] Capsular polysaccharides from serotypes 3, 9V, 14, and 18C can induce better responses when conjugated to diphtheria toxoid than when conjugated to tetanus toxoid [6]. Thus, serotypes 3, 6B, 14, and 18C were conjugated to diphtheria toxoid, and serotypes 1, 4, 5, 7F, 9V, 19F, and 23F were conjugated to tetanus toxoid. Development of this mixed-carrier pneumococcal vaccine was discontinued due in part to technical reasons and the potential for reduced responses when administered with acellular pertussis vaccine [3]. Recently, serotypes 5 and 1 were reported to have one of the lowest OPA titers observed among all 13 pre-natal serotypes, for which there was a relevant correlation between IgG titers and OPA activity [4].It has also been suggested that serotype 3 requires much higher serum IgG concentrations for protection [5]. Summary of the Invention

[0007] The present application provides new and improved mixed carrier multivalent pneumococcal conjugate compositions and vaccines comprising 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 from a different Streptococcus pneumoniae serotype, 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 is selected from CRM 197 or tetanus toxoid, four of the capsular polysaccharides are conjugated to tetanus toxoid, and the remaining capsular polysaccharide is a CRM 197 Two of the four capsular polysaccharides conjugated to tetanus toxoid 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, capsular polysaccharides from serotypes 1, 5, 15B, and 22F are conjugated to tetanus toxoid, and 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 It is complexed with

[0009] In another embodiment of the mixed carrier 21-valent pneumococcal conjugate composition, capsular polysaccharides from serotypes 1, 3, 15B, and 22F are conjugated to tetanus toxoid, and 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 CRM197 is complexed with .

[0010] In yet another embodiment of the mixed carrier 21-valent pneumococcal conjugate composition, capsular polysaccharides from serotypes 3, 5, 15B, and 22F are conjugated to tetanus toxoid, and capsular polysaccharides 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 It is complexed with

[0011] In another aspect, the present application provides 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 conjugated to a capsular polysaccharide from a different serotype of Streptococcus pneumoniae, wherein the Streptococcus pneumoniae serotypes are 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 wherein the protein carrier is a CRMP. 197 or tetanus toxoid, where three of the capsular polysaccharides are conjugated to tetanus toxoid and the remaining capsular polysaccharide is a CRM 197 and three capsular polysaccharides conjugated to tetanus toxoid are selected from the group consisting of serotypes 1, 3, 5, 15B and 22F. In certain embodiments, 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 polysaccharides conjugated to 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 embodiment 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, comprising administering to the subject a prophylactically effective amount of a mixed-carrier 21-valent pneumococcal conjugate composition or a vaccine comprising the same.

[0016] In certain embodiments, the subject is a human who is 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 old 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 old. In other embodiments, the human subject is between 2 and 15 months old or between 6 and 17 years old.

[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 embodiment is an immunogenic conjugate of Streptococcus pneumoniae serotype 9N comprising a serotype 9N capsular glycoside from Streptococcus pneumoniae; and a carrier protein bound to the capsular glycoside, wherein the carrier protein is a CRM 197In certain embodiments of the immunogenic serotype 9N conjugates, mixed carrier multivalent pneumococcal conjugate compositions and vaccines (and methods / uses thereof), the serotype 9N glycoprotein is ligated to a CRMP to form an activated conjugate having a degree of oxidation of 2-19 or 5-10 and a molecular weight of 200-700 kDa. 197 In certain embodiments of the immunogenic serotype 9N conjugates, mixed carrier multivalent pneumococcal conjugate compositions and vaccines (and methods / uses thereof), the immunogenic serotype 9N conjugates may have a molecular weight of 500 to 4,000 kDa.

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

[0021] In certain embodiments of the immunogenic serotype 9N conjugates, mixed carrier multivalent pneumococcal conjugate compositions and vaccines (and methods / uses thereof), 15-60% of the immunogenic serotype 9N conjugates have a K of 0.3 or less on a CL-4B column. d may have:

[0022] In certain embodiments of the immunogenic serotype 9N conjugates, mixed carrier multivalent pneumococcal conjugate compositions and vaccines (and methods / uses thereof), the immunogenic serotype 9N conjugates are prepared using serotype 9N polysaccharides that have been activated to achieve a degree of oxidation of 2 to 19. In certain embodiments, the immunogenic serotype 9N conjugates are prepared using serotype 9N polysaccharides that have been activated to achieve a degree of oxidation of 5 to 10.

[0023] In certain embodiments of the immunogenic serotype 9N conjugate, mixed carrier multivalent pneumococcal conjugate compositions and vaccines (and methods / uses thereof), the Streptococcus pneumoniae serotype 9N glycoprotein is administered in a CRMP by adding 0.02-0.19 μg of periodate per μg of glycoprotein. 197 When complexed with , the complexes have a molecular weight of 500–4,000 kDa and a molecular weight distribution (K d ≦0.3) and may have a glycoside / protein ratio of 0.5 to 2.5.

[0024] In yet another aspect, the present disclosure also provides a method for preparing an immunogenic complex of Streptococcus pneumoniae serotype 9N, comprising: (a) lysing bacterial cells that produce capsular polysaccharide of Streptococcus pneumoniae serotype 9N by fermenting it; (b) purifying capsular glycosides of Streptococcus pneumoniae serotype 9N from lysed 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) Activated glycoconjugates are combined with CRM 197 By mixing with CRM 197 and forming a complex of the capsular glycoside of Streptococcus pneumoniae serotype 9N bound to

[0025] In certain embodiments, the CRM mixed in step (d) 197 may be reacted with a reducing agent to form a complex with activated Streptococcus pneumoniae serotype 9N capsular polysaccharide. In certain embodiments, in step (c), 0.02 to 0.19 μg of periodate is added to 1 μg of Streptococcus pneumoniae serum. The mixture can be reacted with the capsular polysaccharide of strain 9N at 20 to 25°C for 15 to 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 to 900 kDa. 197 The activated Streptococcus pneumoniae serotype 9N capsular polysaccharide admixed with the Streptococcus pneumoniae serotype 9N capsular polysaccharide may have a molecular weight of 200 to 700 kDa. In certain embodiments, the Streptococcus pneumoniae serotype 9N immunogenic complex may have a molecular weight of 500 to 4,000 kDa. In certain embodiments, the CRM against the activated serotype 9N capsular polysaccharide may have a molecular weight of 500 to 4,000 kDa. 197 Initial investment ratio (Carrier CRM 197 In certain embodiments, at least 15-60% of the immunogenic conjugates have a K of 0.3 or less as measured on a CL-4B column. d may have:

[0027] In certain embodiments, the Streptococcus pneumoniae serotype 9N polysaccharide of the present disclosure is purified by adding 0.02 to 0.19 μg of periodate per μg of saccharide to the CRM. 197 When complexed with , the immunogenic complexes have molecular weights of 500–4,000 kDa and 15–60% (K) as measured on a CL-4B column. d ≦0.3) and CRM of 0.5 to 2.5 197 / polysaccharide ratio.

[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] definition In order that this disclosure may be more readily understood, certain terms are first defined below. Additional definitions for these terms, as well as 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 that will become apparent to those skilled in the art upon reading this disclosure and so forth.

[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, including, 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 a stated reference value. In certain embodiments, the term "approximately" or "about," unless otherwise stated or otherwise clear from the context, refers to a range of values ​​that falls 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 or less) of the stated reference value (except where such number would exceed 100% of possible values).

[0033] Conjugate: As used herein and as understood from the appropriate context, the term "conjugate(s)" 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 degree of oxidation of purified or sucrose. The degree of oxidation of a glycoside refers to the number of sugar repeat units per aldehyde group generated when the glycoside is activated with an oxidizing agent. The degree of oxidation of a glycoside can be determined using conventional methods known to those skilled 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 more than one type of protein carrier.

[0037] Multivalent: As used herein, the term "multivalent" refers to a pneumococcal conjugate composition having pneumococcal capsular polysaccharides 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)" refers to a 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 is selected from the group consisting of CRMP, Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F. 197 or tetanus toxoid, in which 1) three of the capsular polysaccharides are conjugated to tetanus toxoid and the remaining capsular polysaccharide is a CRM 197 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 polysaccharide is CRM 197In some embodiments, capsular polysaccharides from serotypes 1, 5, 15B, and 22F are conjugated to tetanus toxoid, and the remaining capsular polysaccharides are from CRM. 197 In another embodiment, capsular polysaccharides from serotypes 1, 3, 15B and 22F are conjugated to tetanus toxoid, and capsular polysaccharides from the remaining serotypes are conjugated to CRM 197 In yet another embodiment, capsular polysaccharides from serotypes 3, 5, 15B and 22F are conjugated to tetanus toxoid, with the remaining capsular polysaccharides being conjugated to CRM. 197 It is complexed with

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

[0040] Pharmaceutically acceptable excipients: Pharmaceutically acceptable excipients useful in this disclosure are conventional. See 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 one or more therapeutic compositions, including vaccines and additional pharmaceutical agents. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, monocalcium phosphate, sorbitol ... Examples of suitable excipients include glycerol stearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. Generally, the nature of the excipient will depend on the particular mode of administration being employed. For example, parenteral formulations usually comprise injectable fluids containing pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, buffer solutions, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid excipients can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered may contain minor 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 dose" refers to an amount or dose required to induce an immune response sufficient to delay the onset of and / or reduce the frequency and / or severity of one or more symptoms caused by infection with Streptococcus pneumoniae.

[0042] Prevention: The term "prevention," as used herein, refers to avoiding signs of disease, delaying the onset, and / or reducing the frequency and / or severity of one or more symptoms of a particular disease, disorder, or condition (e.g., infection with Streptococcus pneumoniae). In some embodiments, prevention is assessed on a population basis such that an agent is considered to provide prevention for 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 susceptible to the disease, disorder, or condition.

[0043] Subject: As used herein, the term "subject" refers to 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." DETAILED DESCRIPTION OF THE INVENTION

[0044] The following description of the disclosed embodiment(s) and examples is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses.

[0045] The present application provides new and improved mixed carrier multivalent pneumococcal conjugate compositions and vaccines containing the same. 197 While CRM has been previously used in single-carrier pneumococcal conjugate vaccines, this application focuses on the use of CRM in mixed-carrier pneumococcal conjugate vaccines. 197 In particular, this application describes the use of CRM as a carrier protein for specific pneumococcal serotypes in multivalent pneumococcal conjugate compositions and vaccines. 197 and tetanus toxoid.

[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 the previous finding that serotype 3 is more immunogenic when conjugated to diphtheria toxoid rather than tetanus toxoid [6]. This application also describes the successful conjugation of serotypes 1, 5, 15B, and 22F to tetanus toxoid as part of a mixed carrier vaccine. Furthermore, antibody responses to serotype 3 conjugated to tetanus toxoid in a mixed carrier multivalent, e.g., 21-valent, pneumococcal conjugate composition are significantly higher than those observed in a single-carrier, 13-valent pneumococcal conjugate composition (PREVNAR13). 197They also disclosed the unexpected finding that the activity of the ATP-binding protein was approximately 4.5 times higher than when the ATP-binding protein was complexed with ATP.

[0047] Furthermore, this unexpected finding was not limited to serotype 3, but was also observed for other serotypes conjugated to tetanus toxoid in a mixed carrier multivalent pneumococcal conjugate composition. For example, as shown in the Examples, conjugation of serotypes 1 and 5 or 3 and 5 to tetanus toxoid in a mixed carrier pneumococcal conjugate composition, with the remaining serotypes being CRM 197 Those conjugated to a single carrier (e.g., PCV21(1 / 5 / 15B / 22F)-TT and PCV21(3 / 5 / 15B / 22F)-TT) are CRMs in a single carrier, pneumococcal conjugate composition (PREVNAR13). 197 Consistently, tetanus toxoid-conjugated serotypes induced significantly enhanced antibody responses compared with antibody responses (IgG responses or MOPA titers) to the same serotypes conjugated to tetanus toxoid.

[0048] Tetanus toxoid is a CRM 197 Therefore, conjugating three or four of serotypes 1, 3, 5, 15B, and 22F to tetanus toxoid as part of a combined carrier vaccine is a promising option for CRM 197 This results in a reduced polysaccharide to carrier ("PS / C") ratio for those serotypes conjugated to tetanus toxoid, compared to the PS / C ratio for those same serotypes conjugated to a single carrier smaller than tetanus toxoid, such as 15B, 22F, 35B, 36B, 37B, 38B, 39B, 40B, 41B, 42B, 43B, 44B, 45B, 46B, 47B, 48B, 49B, 50B, 51B, 52B, 53B, 54B, 55B, 56B, 57B, 58B, 59B, 60B, 61B, 62B, 63B, 64B, 65B, 66B, 67B, 68B, 69B, 70B, 71B, 72B, 73B, 74B, 75B, 76B, 77B, 78B, 79B, 80B, 81B, 82B, 83B, 84B, 85B, 86B, 87B, 88B, 89B, 90B, 91B, 92B, 93B, 94B, 95B, 96B, 97B, 98B, 99B, 100B, 101B, 102B, 103B, 104B, 105B, 106B, 107B, 108B, 110B, 111B, 112B, 113B, 114B, 115B, 116B, 117B, 11

[0049] The mixed carrier 21-valent pneumococcal conjugate compositions described in this application also include pneumococcal serotypes not currently covered by the three pneumococcal conjugate vaccines currently available on the global market: PREVNAR (called Prevenar in some countries), SYNFLORIX, and PREVNAR 13. Disease caused by pneumococcal serotypes not currently covered is increasing due in part to the development of antimicrobial resistance, an increasing number of immunocompromised patients, and a lack of immune pressure. For example, none of the currently available pneumococcal conjugate vaccines includes serotype 9N. Also, none of the currently available pneumococcal conjugate vaccines includes serotypes 8, 10A, 11A, 12F, 15B, 22F, and 33F. The present disclosure provides a mixed carrier (tetanus toxoid and CRM) for serotypes 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F. 197 ) demonstrated the successful implementation of a pneumococcal conjugate vaccine and that serotype 9N induced approximately 40-50 times higher antibody responses than PREVNAR13.

[0050] Pneumococcus polysaccharide serotype 9N The serotype 9N polysaccharide can be obtained directly from the bacteria using isolation procedures known to those skilled in the art, including but not limited to the methods disclosed in U.S. Patent Application Publication No. 2006 / 0228380. Alternatively, the glycoside can be produced using synthetic protocols.

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

[0052] The bacterial cells are typically grown in a medium such as a soy-based medium. Following fermentation of the bacterial cells to produce the capsular polysaccharide of Streptococcus pneumoniae serotype 9N, the bacterial cells are lysed to produce a cell lysate, which is then purified by centrifugation, depth filtration, sedimentation, ultrafiltration, treatment with activated carbon, diafiltration, and / or column chromatography (as disclosed in U.S. Patent Application Publication No. 2006 / 0228380). The serotype 9N polysaccharide can be isolated from the cell lysate using purification techniques known in the art, including but not limited to, methods such as ELISA and immunoassay. 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 some embodiments, the purified polysaccharide purified from Streptococcus pneumoniae serotype 9N prior to conjugation has a molecular weight of 5 to 5,000 kDa. In some embodiments, the serotype 9N capsular polysaccharide prior to conjugation has a molecular weight of 50 to 1,000 kDa. In some embodiments, the serotype 9N capsular polysaccharide prior to conjugation has a molecular weight of 70 to 900 kDa. In some embodiments, the serotype 9N capsular polysaccharide prior to conjugation has a molecular weight of 100 to 800 kDa. In certain embodiments, purified serotype 9N capsular polysaccharide may be activated prior to conjugation to have a molecular weight of 50-800 kDa, 80-780 kDa, 100-770 kDa, 120-760 kDa, 140-750 kDa, 150-740 kDa, 160-730 kDa, 170-735 kDa, 180-720 kDa, 190-710 kDa, 200-700 kDa, 220-690 kDa, 240-680 kDa, 260-670 kDa, 270-660 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 may be characterized by its degree of oxidation and molecular weight. In certain embodiments, the activated serotype 9N polysaccharide may have a degree of oxidation of 0.5 to 25, 0.6 to 23, 0.8 to 21, 1 to 20.8, 1.1 to 20.5, 1.2 to 20.3, 1.3 to 20, 1.4 to 19.5, 1.5 to 19.3, 1.6 to 19.2, 1.7 to 19.1, 2 to 19, 3 to 18, 4 to 15, or 5 to 10.

[0055] Polysaccharides may be slightly reduced in size during normal purification procedures. Also, as described in this disclosure, polysaccharides may be subjected to sizing before conjugation. The molecular weight ranges listed above refer to purified polysaccharides after a final sizing step (e.g., after purification, hydrolysis, and activation) before conjugation.

[0056] Mixed-carrier polyvalent pneumococcal complex composition and method of manufacturing the same The present 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 is a CRMP. 197 or tetanus toxoid, where 3-4 of the capsular polysaccharides are conjugated to tetanus toxoid and the remaining capsular polysaccharides are CRM 197 and three to four capsular polysaccharides conjugated to tetanus toxoid are selected from the group consisting of serotypes 1, 3, 5, 15B, and 22F. In certain embodiments, three of the capsular polysaccharides are conjugated to tetanus toxoid and the remaining capsular polysaccharides are conjugated to CRM. 197In certain embodiments, four of the capsular polysaccharides are conjugated to tetanus toxoid, and the remaining capsular polysaccharide is conjugated to CRM 197 It is complexed with

[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 The capsular polysaccharide-protein conjugate comprises a protein carrier conjugated to capsular polysaccharide from different serotypes 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 is CRM 197 or tetanus toxoid, four of the capsular polysaccharides are conjugated to tetanus toxoid, and the remaining capsular polysaccharide is a CRM 197 and two of the four capsular polysaccharides conjugated to tetanus toxoid are selected from the group consisting of serotypes 1, 3, and 5, and the remaining two capsular polysaccharides are serotypes 15B and 22F.

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

[0059] In polysaccharide-protein conjugate vaccines, a carrier protein is conjugated to a polysaccharide antigen primarily to help enhance the immune response (e.g., antibody response) to the polysaccharide antigen. The carrier protein is preferably a non-toxic protein. The carrier protein should be amenable to conjugation with pneumococcal polysaccharides using standard conjugation procedures, as discussed in more detail below. Carrier proteins used in mixed-carrier 21-valent pneumococcal conjugate compositions include tetanus toxoid (TT) and CRM. 197 and each of these has been used in the design of pneumococcal conjugate vaccines, but never in the same mixed carrier vaccine.

[0060] CRM 197 CRM is a non-toxic variant of diphtheria toxin (i.e., a toxoid) that retains the immunological properties of wild-type diphtheria toxin. 197 CRM differs from wild-type diphtheria toxin by a single base in the structural gene, resulting in a single amino acid substitution from glutamic acid to glycine. 197 CRM is typically isolated from cultures of Corynebacterium diphtheria strain C7 (β197) grown in a medium based on casamino acids and yeast extract. 197 CRM can be purified by ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography. 197 CRM can be prepared recombinantly according to U.S. Patent No. 5,614,382, which is incorporated herein by reference in its entirety. 197 has been used in the design of pneumococcal conjugate vaccines but has never been used as part of a combined 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 in cultures of Clostridium tetani. Tetanus toxin is an approximately 150 kDa protein consisting 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, e.g., [7], [8]) or chromatographic techniques (e.g., as disclosed in WO 1996 / 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, the CRM in mixed-carrier pneumococcal conjugate vaccines 197 The use of tetanus toxoid in combination with diphtheria toxoid is novel. The art also teaches away from conjugating serotype 3 to tetanus toxoid in mixed carrier pneumococcal conjugate vaccines because serotype 3 has been shown to be more immunogenic when conjugated to diphtheria toxoid compared to tetanus toxoid.[6]

[0063] Pneumococcal capsular polysaccharides for use in the compositions and vaccines described herein, including capsular polysaccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, are described, for example, in WO2006 / 110381, WO2008 / 118752, WO2006 / 110352, and U.S. Patent Application Publication No. 2006 / 022 Pneumococcal capsular polysaccharides can be prepared from Streptococcus pneumoniae using any available technique, including standard techniques known to those of skill in the art, including those disclosed in US Pat. Nos. 8380, 2006 / 0228381, 2007 / 0184071, 2007 / 0184072, 2007 / 0231340, 2008 / 0102498, and 2008 / 0286838 (all of which are incorporated by reference in their entireties). For example, each pneumococcal capsular polysaccharide serotype can be grown in culture medium (e.g., soy-based medium). Cells can be lysed, and individual polysaccharides can be purified from the lysate by centrifugation, precipitation, ultrafiltration, and / or column chromatography. Pneumococcal capsular polysaccharides can also be produced using synthetic protocols.

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

[0065] Pneumococcal conjugates of each serotype can be prepared by conjugating the capsular polysaccharide of each serotype to a carrier protein. The different pneumococcal conjugates can be formulated into compositions that comprise a single dosage formulation.

[0066] To prepare polysaccharide-protein conjugates, capsular polysaccharides prepared from each pneumococcal serotype can be chemically activated to render them reactive with carrier proteins. Once activated, each capsular polysaccharide can be separately conjugated to a carrier protein to form a glycoconjugate. Chemical activation of the polysaccharides and subsequent conjugation to the carrier protein can be accomplished by conventional methods. For example, the vicinal hydroxyl groups at the termini of capsular polysaccharides can be oxidized to aldehyde groups by an oxidizing agent such as periodate (including sodium periodate, potassium periodate, or periodic acid), as disclosed in U.S. Pat. Nos. 4,365,170, 4,673,574, and 4,902,506 (incorporated herein by reference in their entireties). Periodate randomly oxidizes the vicinal hydroxyl groups of carbohydrates to form reactive aldehyde groups, resulting in C-C bond cleavage. The term "periodate" includes both periodate and periodic acid. The term also includes metaperiodate (IO 4- ) and orthoperiodate (IO 65- The term "periodate" also includes 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, periodate may be used in an amount of about 0.03-0.17 μg per μg of polysaccharide. In certain embodiments, periodate may be used in an amount of about 0.025-0.18 μg or about 0.02-0.19 μg per μg of polysaccharide. The glycoside may be activated as desired within the above range. Outside of this range, the effect may be insufficient.

[0068] Polysaccharides can also be activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form cyanate esters, which are then coupled to amino groups on a carrier protein either directly or via a spacer or linker group.

[0069] For example, the spacer can be cystamine or cysteamine to provide a thiolated polysaccharide that can be attached to a carrier via a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[y-maleimidobutyloxy]succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g., using iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl) aminobenzoate (SlAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA), or succinimidyl 3-[bromoacetamido]propionate (SBAP)). Preferably, cyanate esters (optionally prepared by COAP chemistry) are coupled with hexanediamine or adipic acid dihydrazide (AOH), and amino-derivatized glycosides are conjugated to carrier proteins using carbodiimide (e.g., EDAC or EDC) chemistry via carboxyl groups on the protein carrier. Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348, and WO 96 / 129094, all of which are incorporated herein by reference in their entireties.

[0070] Conjugation of the activated capsular polysaccharide to a carrier protein can be achieved, for example, by reductive amination, as described, for example, in U.S. Patent Application Publication Nos. 2006 / 0228380, 2007 / 0231340, 2007 / 0184071, and 2007 / 0184072, WO 2006 / 110381, WO 2008 / 079653, and WO 2008 / 143709 (all of which are incorporated by reference in their entireties). For example, the activated capsular polysaccharide and the carrier protein can be reacted with a reducing agent to form the conjugate. Suitable reducing agents include borohydrides, e.g., sodium cyanoborohydride, borane-pyridine, sodium triacetoxyborohydride, sodium borohydride, or borohydride ion exchange resins. At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the complex. The unreacted aldehyde groups can be capped using a suitable capping agent, such as sodium borohydride (NaBH). 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 DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent. Other possible reducing agents include, but are not limited to, amine-boranes, such as pyridine-borane, 2-picoline-borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH, benzylamine-BH, or 5-ethyl-2-methylpyridine-borane (PEMB).

[0071] The activated capsular polysaccharide can be conjugated directly to a carrier protein or indirectly through the use of a spacer or linker, e.g., a bifunctional linker. The linker is optionally heterobifunctional or homobifunctional, e.g., having a reactive amino group and a reactive It has one reactive carboxylic acid group, two reactive amino groups or two reactive carboxylic acid groups.

[0072] Other suitable techniques for conjugation use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU, as described, for example, in International Patent Application Publication WO 98 / 42721 (incorporated by reference in its entirety). Conjugation can involve a carbonyl linker, which can be formed by reaction of a free hydroxyl group of a glycoside with 1,1'-carbonyldiimidazole (CD1) (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 may involve reduction of the anomeric terminus to a primary hydroxyl group, optional protection / deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling of the CDI carbamate intermediate to an amino group on the protein.

[0073] The polysaccharide to carrier protein ratio for pneumococcal conjugate vaccines typically ranges from 0.3 to 3.0 (w / w), but may vary depending on the serotype. The ratio can be determined by independent measurement of the amount of protein and polysaccharide present, or by methods known in the art that provide a direct measurement of the ratio. 1 Methods including H NMR spectroscopy or SEC-HPLC-UV / RI with dual monitoring (e.g., refractive index and UV (for total material and protein content, respectively)) can profile the glycoside / protein ratio across the size distribution of complexes, as can SEC-HPLC-MALLS or MALDI-TOF-MS.

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

[0075] Formulation of vaccine compositions can be accomplished using art-recognized methods. Vaccine compositions are formulated to be compatible with their intended route of administration. Individual pneumococcal capsular polysaccharide-protein complexes can be formulated with a physiologically acceptable vehicle to prepare the composition. Examples of such vehicles include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and dextrose solution.

[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 (alum, aluminum salts such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, aluminum hydroxyphosphate sulfate, etc.) to which the antigen is adsorbed, or water-in-oil emulsions in which an antigen solution is emulsified in mineral oil (e.g., Freund's incomplete adjuvant), sometimes including 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. Patent 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: , 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 about 0.1 mg to 2.5 mg of aluminum-based adjuvant. In other embodiments, a single 0.5 ml vaccine dose is formulated to contain 0.1 mg to 2 mg, 0.1 mg to 1 mg, 0.1 mg to 0.5 mg, 0.1 mg to 0.2 mg, 0.125 mg to 2.5 mg, 0.125 mg to 0.5 mg, 0.125 mg to 0.2 mg, or 0.125 to 0.25 mg of aluminum-based adjuvant. In certain embodiments, a single 0.5 ml vaccine dose is formulated to contain about 0.125 mg to about 0.250 mg of aluminum-based adjuvant. In certain embodiments, a single 0.5 ml vaccine dose is formulated to contain about 0.125 mg of aluminum-based adjuvant. In certain embodiments, a single 0.5 ml vaccine dose is formulated to contain about 0.250 mg of 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 a particular embodiment, the adjuvant is aluminum phosphate.

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

[0081] Characterization of pneumococcal capsular polysaccharide-protein carrier complexes In certain embodiments, the polysaccharide-protein carrier conjugate may have a molecular weight between 100 and 10,000 kDa. In certain embodiments, the conjugate has a molecular weight between 200 and 9,000 kDa. In certain embodiments, the conjugate has a molecular weight between 300 and 8,000 kDa. In certain embodiments, the conjugate has a molecular weight between 400 and 7,000 kDa. In certain embodiments, the conjugate has a molecular weight between 500 and 6,000 kDa. In certain embodiments, the conjugate has a molecular weight between 600 and 5,000 kDa. In certain embodiments, the conjugate has a molecular weight between 500 and 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 conjugate can be stably formed in high yield, the proportion of free polysaccharides can be reduced, and excellent immunogenicity can be obtained within the above molecular weight range.

[0083] After the individual polysaccharide-protein conjugates are purified, they are combined to formulate the immunogenic compositions of the present disclosure.

[0084] The glyco-protein conjugates of the serotypes of the present disclosure may be characterized by the ratio of polysaccharide to protein carrier (amount of polysaccharide / amount of protein carrier, w / w).

[0085] In certain embodiments, the ratio of polysaccharide to protein carrier (w / w) in the polysaccharide-protein carrier conjugate for each serotype is between 0.5 and 2.5, between 0.4 and 2.3, between 0.3 and 2.1, between 0.24 and 2, between 0.2 and 1.8, between 0.18 and 1.6, between 0.16 and 1.4, between 0.14 and 1.2, between 0.12 and 1, or between 0.1 and 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 polysaccharide to protein carrier is within the above range, the conjugate can be stably formed with high yield, the proportion of free polysaccharide can be reduced, excellent immunogenicity can be achieved, and the conjugate can be stably maintained within the above range without interference from other serotypes.

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

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

[0089] The polysaccharide-protein carrier conjugates of each serotype also exhibit a molecular size distribution (K d) can be characterized. To determine the relative molecular size distribution of the complexes, size exclusion chromatography media (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 complexes. Large molecules excluded from the pores in the media elute faster than small molecules. A fraction collector is used to collect the column eluate. Fractions are tested colorimetrically by glycoside assay. K d For the determination of K, the column is divided into two fractions (V0; K) where the molecule is completely excluded. d = 0) and the fraction representing the maximum retention (V i ;K d = 1). The fraction (V e ) is the formula K d =(V e -V0) / (V i -V0) d Related to.

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

[0091] In certain embodiments, at least 20% of the polysaccharide-protein carrier conjugates of each serotype have a K of 0.3 or less on a CL-4B column. d 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 conjugates of each serotype may have a K of 0.3 or less on a CL-4B column. d In certain embodiments, at least 60% of the polysaccharide-protein carrier conjugates of each serotype may have a K of 0.3 or less on a CL-4B column. dIn certain embodiments, at least 50-80% of the polysaccharide-protein carrier conjugates of each serotype may have a K of 0.3 or less on a CL-4B column. d In certain embodiments, at least 65-80% of the polysaccharide-protein carrier conjugates of each serotype may have a K of 0.3 or less on a CL-4B column. d In certain embodiments, at least 15-60% of the glycoprotein conjugates of each serotype may have a K of 0.3 or less on a CL-4B column. d may have:

[0092] Prophylactic Methods and Uses In one aspect, the 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 comprises at least a buffer, such as succinate buffer, a salt, such as sodium chloride, and / or a surfactant, such as a polyoxyethylene sorbitan ester (e.g., polysorbate 80). In some embodiments, three or four capsular polysaccharides from a particular serotype are conjugated to tetanus toxoid, as described above, 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 It is complexed to (21-valent).

[0093] In one embodiment, capsular polysaccharides from serotypes 1, 5, 15B and 22F are conjugated to tetanus toxoid, and 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 It is complexed to (21-valent).

[0094] In another embodiment, capsular polysaccharides from serotypes 1, 3, 15B and 22F are conjugated to tetanus toxoid and 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 is complexed to (21-valent).

[0095] In yet another embodiment, capsular polysaccharides from serotypes 3, 5, 15B and 22F are conjugated to tetanus toxoid and capsular polysaccharides 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 It is complexed to (21-valent).

[0096] In some embodiments, the vaccine elicits a protective immune response in a human subject against disease 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, 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 may be administered by any route, including, for example, systemically or mucosally, as described in further detail below.

[0098] In some embodiments, the human subject is an elderly subject and the disease is pneumonia or invasive pneumococcal disease (IPD). In some 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 some embodiments, the infant is 0-2 years old. In other embodiments, the infant is 2-15 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 conjugate or prophylactically effective amount of the mixed-carrier multivalent pneumococcal conjugate composition in each vaccine dose can be selected to induce protection without significant side effects. Such amounts can vary depending on the serotype of pneumococcus. Generally, each dose can contain about 0.1 μg to about 100 μg, specifically about 0.1 to 10 μg, and more specifically about 1 μg to about 5 μg of polysaccharide. Optimal amounts of components for a particular vaccine can be elucidated through standard studies involving observation of appropriate immune responses in subjects. For example, amounts for vaccination of human subjects can be determined by extrapolating the results of animal studies. Alternatively, doses can be determined empirically.

[0102] In some embodiments, the vaccine or mixed carrier 21-valent pneumococcal conjugate composition contains 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 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 contains about 2 μg to about 2.5 μg of each capsular polysaccharide, except for serotype 6B and optionally serotype 3, present in an amount of about 4 μg to about 5 μg; about 2 μg to about 25 μg of TT; about 40 μg to about 75 μg of CRM. 197 and optionally a single 0.5 ml dose formulated to contain 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 may 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 may be a single 0.5 ml dose formulated to contain about 2 μg to about 2.5 μg of each of capsular polysaccharides, excluding up to six 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 six 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 may be a single 0.5 ml dose formulated to contain about 2.2 μg of each of capsular polysaccharides, excluding up to six 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 six 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 The capsular polysaccharides may be a single 0.5 ml dose formulated to contain about 2 μg to about 2.5 μg of serotypes 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 22F, 23F, and 33F capsular polysaccharides and about 4 μg to about 5 μg of serotypes 1, 3, 6B, 19A, and 19F capsular polysaccharides.

[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 to 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 to 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 to about 2.5 μg of serotype 1, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F capsular polysaccharides and about 4 to about 5 μg of serotype 6B capsular polysaccharides and about 8 to about 9 μg of serotype 3 capsular polysaccharides, more preferably about 8.8 μg of serotype 3 capsular polysaccharides.

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

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

[0111] In some embodiments, the mixed carrier 21-valent pneumococcal conjugate composition comprises pneumococcal capsular polysaccharides from serotypes 3, 5, 15B, and 22F each conjugated to TT, and capsular polysaccharides 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 In one embodiment, each 0.5 mL dose contains about 2.2 μg of each capsular polysaccharide except for about 4.4 μg of serotype 6B, about 2 μg to about 25 μg of TT carrier protein (for serotypes 3, 5, 15B, and 22F only), and about 40 μg to about 70 μg of CRM. 197 It may 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 aluminum phosphate) adjuvant, and sodium chloride and sodium succinate buffer as excipients. In another embodiment, each 0.5 mL dose contains about 4.4 μg of capsular polysaccharides of up to six species selected from the group consisting of serotypes 1, 3, 4, 5, 6B, 9V, 19A, and 19F. Approximately 2.2 μg of each capsular polysaccharide excluding the above, 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 197The vaccine may 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 aluminum phosphate) adjuvant, and sodium chloride and sodium succinate buffer as excipients. In one embodiment, about 4.4 μg of up to six capsular polysaccharides 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, about 4.4 μg, about 2 μg to about 25 μg of TT carrier protein (for serotypes 3, 5, 15B, and 22F only), and about 40 μg to about 70 μg of CRM. 197 It may 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 aluminum phosphate) adjuvant, and sodium chloride and sodium succinate buffer as excipients.

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

[0113] In some embodiments, the liquid formulation can be filled into single-dose syringes without preservatives. After shaking, the liquid formulation becomes a vaccine that is a homogenous white suspension ready 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 two, three, four, or more times at appropriate intervals, e.g., 1, 2, 3, 4, 5, or 6 months apart, or a combination thereof. In some embodiments, the mixed carrier 21-valent pneumococcal conjugate composition is administered to an infant four times within the first 15 months of life, including, for example, at about 2, 3, 4, and 12-15 months of age, about 3, 4, 5, and 12-15 months of age, or 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 an infant three 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 may also include one or more proteins from Streptococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for inclusion include those identified in International Patent Application WO 02 / 083855 and those described in International Patent Application WO 02 / 053761.

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

[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 liquid or lyophilized form. In some embodiments, the injectable compositions are prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. In some embodiments, 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 discussed, for example, in Remington's Pharmaceutical Sciences, 1999. thed., 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 metered 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. Pat. Nos. 4,886,499, 5,190,521, 5,328,483, 5,527,288, 4,270,537, 5,015,235, 5,141,496, and 5,417,662 (all of which are incorporated herein by reference). Intradermal compositions can also be administered by devices that limit the effective penetration length of a needle into the skin, such as those described in WO1999 / 34850, which is incorporated herein by reference, and their functional equivalents.Also suitable are jet injection devices that deliver liquid vaccines to the dermis through a liquid jet injector or through 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 ... Nos. 5,312,335, 5,503,627, 5,064,413, 5,520,639, 4,596,556, 4,790,824, 4,941,880, 4,940,460, WO1997 / 37705, and WO1997 / 13537, all of which are incorporated herein by reference.Also suitable are ballistic powder / particle delivery devices that use compressed gas to accelerate the vaccine in powder form through the outer layer of the skin and into the dermis, and conventional syringes can be used in the classical Mantoux technique 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 injectable organic esters such as ethyl oleate. Examples of oils include vegetable or animal oils, synthetic oils such as peanut oil, soybean oil, olive oil, sunflower oil, cod liver oil, and marine oil, and lipids derived from milk or eggs. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases.

[0119] The mixed carrier 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, compositions for infusion or injection are preferably essentially isotonic. Therefore, isotonicity or hypertonicity may be advantageous for the storage of the composition. If the composition is hypertonic, it can be diluted to isotonicity before administration. The tonicity agent can be an ionic agent such as a salt or a non-ionic agent such as a carbohydrate. Ionic tonicity agents include, but are not limited to, sodium chloride, calcium chloride, potassium chloride, and magnesium chloride. Non-ionic tonicity 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 solution having a buffering capacity of pH 4 to pH 10, e.g., pH 5 to pH 9, or pH 6 to pH 8. The buffer solution may be selected from those suitable for the United States Pharmacopeia (USP). For example, the buffer solution may 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 mixed carrier 21-valent pneumococcal conjugate composition may include a surfactant. Examples of surfactants include, but are not limited to, polyoxyethylene sorbitan esters (commonly referred to as Tweens), particularly polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and butylene oxide (BO) (such as DOWFAX); octoxynols with different repeating ethoxy (oxy-1,2-ethanediyl) groups, particularly 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, and oleyl alcohols (Brij surfactants), particularly triethylene glycol monolauryl ether (Brij 30); and sorbitan ethers known as SPAN®, particularly sorbitan trioleate (Span 85) and sorbitan monolaurate.

[0121] A mixture of surfactants such as Tween 80 / Span 85 can be used. A combination of a polyoxyethylene sorbitan ester such as Tween 80 with an octoxynol such as Triton X-100 is also suitable. A combination of Laureth 9 with a Tween and / or an octoxynol is also advantageous. Preferably, the amount of polyoxyethylene sorbitan ester (such as Tween 80) included may be 0.01% to 1% (w / v), 0.01% to 0.1% (w / v), 0.01% to 0.05% (w / v), or about 0.02%; the amount of octylphenoxypolyoxyethanol or nonylphenoxypolyoxyethanol (such as Triton X-100) included may be 0.001% to 0.1% (w / v), particularly 0.005% to 0.02%; and the amount of polyoxyethylene ether (such as Laureth 9) included may be 0.1% to 20% (w / v), optionally 0.1% to 10%, particularly 0.1% to 1% or about 0.5%.

[0122] In some embodiments, the mixed carrier 21-valent pneumococcal conjugate composition can be delivered via a controlled release system. For example, intravenous infusion, transdermal patch, liposomes, or other routes of administration can be used. 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 reference to the following specific examples. These examples are included for illustrative purposes only and are not intended to limit the scope of the invention. [Example]

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

[0125] Cultivation of S. pneumoniae and purification of capsular polysaccharides 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 No. 6301; serotype 3: ATCC No. 6303; serotype 4: ATCC No. 6304; serotype 5: ATCC No. 6305; serotype 6A: ATCC No. 6306; serotype 6B: ATCC No. 6326; serotype 7F: ATCC No. 10351; serotype 9N: ATCC No. 6309; serotype 9V: ATCC No. 10368; serotype 14: ATCC No. 6314; serotype 18C: ATCC No. 10356; serotype 19A: ATCC No. 10357; serotype 19F: ATCC No. 6319; serotype 23F: ATCC No. 6323). Internal 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 as a encapsulated, motile, Gram-positive, lancet diplococci, and alpha-hemolytic on blood agar. Serotypes were identified by the Quelling test (U.S. Patent No. 5,847,112) using specific antisera.

[0126] Preparation of cell banks

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

[0128] Cultivation and collection

[0129] Cultures from the working cell bank were inoculated into seed bottles containing soy-based medium and cultivated. After reaching a target optical density (absorbance), the seed bottles were used to inoculate fermenters containing soy-based medium. The cultivation was terminated when the optical density value began to remain constant. After cultivation was terminated, sodium deoxycholate was added to the culture to lyse the cells. The resulting contents of the fermenter were cooled to induce protein precipitation. The mixture was then centrifuged to remove precipitated proteins and cell debris.

[0130] purification

[0131] The solution obtained from the centrifugation was filtered through a depth filter to remove proteins and cell debris that were not precipitated by 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 diafiltered with approximately 10 volumes of triple-distilled water. The concentrate was sterile filtered and stored at -20°C.

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

[0133] The polysaccharides of different serotypes are activated according to different pathways and then transported to the carrier protein CRM. 197 Specifically, each of the capsular polysaccharides of all serotypes except 15B and 22F was conjugated to CRM 197 Conjugates were prepared by conjugating each of the capsular polysaccharides of serotypes 1, 3, 5, 15B, and 22F to TT. Depending on the size of the native serotype, the activation process may involve reducing the size of each capsular polysaccharide to a target molecular weight, chemical activation, and buffer exchange via ultrafiltration. The conjugates 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 conjugating polymers in which an amide bond is formed between a primary amine (-NH2) group on a protein and an aldehyde on a sugar. Aldehyde groups are added to pneumococcal capsular polysaccharides to facilitate conjugation to carrier proteins. The vicinal diol structure of the monosaccharide can be oxidized with sodium periodate (NaIO4) to form aldehyde groups. 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 (0.05 M final base concentration) was added to a solution of capsular polysaccharide, and the solution was incubated at 50±2° C. The solution was then cooled to a temperature in the range of about 21° C. to about 25° C., and hydrochloric acid was added to a final pH of 6.0±0.1, thereby terminating the hydrolysis.

[0138] For serotypes 3, 8, 11A, and 15B, hydrochloric acid (0.01 M final acid concentration) was added to the capsular polysaccharide solution, 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 to a final pH of 6.0±0.1, thereby stopping the hydrolysis.

[0139] For serotype 4, hydrochloric acid (0.1 M final acid concentration) was added to a solution of capsular polysaccharide, and the solution was incubated at 45±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 to a final pH of 6.0±0.1, thereby stopping the hydrolysis.

[0140] For serotype 6A, glacial acetic acid (0.1 M final acid concentration) was added to a 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 1 M sodium hydroxide was added to a final pH of 6.0±0.1, thereby terminating the hydrolysis.

[0141] For serotype 12F, hydrochloric acid (0.01 M final acid concentration) was added to the capsular polysaccharide solution, and the solution was incubated at 70 ± 2°C. The solution was then cooled to a temperature ranging from about 21°C to about 25°C, and 0.1 M sodium phosphate was added to give a solution with a final pH of 6.0 ± 0.1. This stopped the hydrolysis.

[0142] For serotypes 14 and 18C, glacial acetic acid (0.2 M final acid concentration) was added to the capsular polysaccharide solution, 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 to the solution to a final pH of 6.0 ± 0.1, thereby stopping the hydrolysis.

[0143] For serotypes 22F and 33F, hydrochloric acid (0.01 M final acid concentration) was added to the capsular polysaccharide solution, 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 to a final pH of 6.0±0.1, thereby stopping the hydrolysis.

[0144] Each of the resulting 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 activation of each pneumococcal glycoside was determined based on the molar mass of the repeating unit. After thorough mixing, the oxidation reaction proceeded for 16-20 hours at 21-25°C for all serotypes except 1, 7F, and 19F, with the temperature kept below 10°C. To help maintain consistent and stable production of the conjugates, a range of degree of oxidation (Do) levels for each serotype was targeted during the conjugation process. Preferred target ranges for Do levels for each serotype are shown in Tables 1 and 2. [Table 1] [Table 2]

[0147] Step 3: Ultrafiltration

[0148] The oxidized glycosides were concentrated and diafiltered with WFI through a 100 kDa MWCO ultrafilter (a 30 kDa ultrafilter for serotype 1 and a 5 kDa ultrafilter for serotype 18C). Diafiltration was performed using 0.9% sodium chloride solution for serotype 1, 0.01 M sodium acetate buffer (pH 4.5) for serotypes 7F and 23F, and 0.01 M sodium phosphate buffer (pH 6.0) for serotype 19F. The permeate was discarded, and the retentate was filtered through a 0.2 μm filter.

[0149] Step 4: Freeze drying

[0150] For capsular polysaccharides of serotypes 3, 4, 5, 8, 9N, 9V, 10A, 14, and 33F that are conjugated to carrier proteins by using aqueous solvents, mixed solutions of polysaccharide and carrier protein were prepared without adding additional sucrose, lyophilized, and then stored at −25°C ± 5°C.

[0151] For the capsular polysaccharides of serotypes 1 and 18C that were conjugated to carrier proteins by using aqueous solvents, the polysaccharides and carrier proteins were prepared independently without the addition of additional sucrose, lyophilized, and then stored at −25°C ± 5°C.

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

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

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

[0155] (2) Complexation process

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

[0157] Step 1: Melting

[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 to reaction concentrates at a set ratio for each serotype using sodium phosphate buffer at 23 ± 2°C.

[0160] Dimethyl sulfoxide (DMSO) complexation

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

[0162] Step 2: Conjugation reaction

[0163] Aqueous complexation

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

[0165] The reaction mixture was incubated at 23°C to 37°C for 44 to 106 hours. The reaction temperature and time were adjusted depending on 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 provide 1.8 to 2.2 molar equivalents of sodium borohydride per mole of glycoconjugate. The mixture was incubated at 23±2°C for 3 to 6 hours. This procedure reduced the amount of unreacted aldehyde present on the glycoconjugate. The mixture was then diluted with 0.9% sodium chloride, and the diluted conjugation mixture was filtered using a 0.8 or 0.45 μm prefilter.

[0166] DMSO conjugation

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

[0168] Step 3: Ultrafiltration

[0169] The diluted conjugate mixture was concentrated and diafiltered with a minimum of 15 volumes of 0.9% sodium chloride or buffer through a 100 kDa MWCO or 300 kDa MWCO ultrafilter, and the composition and pH of the buffer used in the process varied depending on the serotype.

[0170] Step 4: Sterile filtration

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

[0172] Example 3. Formulation of a multivalent pneumococcal conjugate vaccine

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

[0174] PCV21(1 / 5 / 15B / 22F)-TT conjugates the polysaccharides of serotypes 1, 5, 15B, and 22F to TT, and the polysaccharides of serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, and 33F to CRM. 197 The polysaccharide-conjugates were prepared by conjugating the polysaccharide to

[0175] PCV21(3 / 5 / 15B / 22F)-TT conjugates the polysaccharides of serotypes 3, 5, 15B, and 22F to TT, and the polysaccharides of serotypes 1, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, and 33F to CRM. 197 The polysaccharide-conjugates were prepared by conjugating the polysaccharide to

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

[0177] A 0.5 ml total dose of the 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 to 25 μg of TT (for serotypes 3, 5, 15B, and 22F), and 0.250 mg of elemental aluminum (1.13 mg aluminum phosphate), with other components and their contents identical to those of PCV21(1 / 5 / 15B / 22F)-TT.

[0178] Example 4. Immunogenicity of multivalent pneumococcal conjugate vaccines The mixed-carrier polyvalent pneumococcal vaccines, PCV21(1 / 5 / 15B / 22F)-TT and PCV21(3 / 5 / 15B / 22F)-TT, prepared in Example 3, were tested for their ability to induce immunogenic responses in rabbits. Immunogenicity was assessed by antigen-specific ELISA for serum IgG concentration and opsonophagocytosis assay (OPA) for antibody functionality. New Zealand White rabbits were immunized intramuscularly at weeks 0 and 2 with a dose of each polysaccharide 5% higher than the planned human clinical dose in the formulation (2.31 μg of each polysaccharide, except for 6B, which was 4.62 μg) or with the human dose (2.2 μg of each polysaccharide, except for 6B, which was 4.4 μg). Serum was collected every two weeks after immunization. Both concentrations showed similar results.

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

[0180] Serotype-specific IgG concentration measurement

[0181] Capsular polysaccharide (PnP) for each serotype was coated onto 96-well plates at 0.5 μg / well to 1 μg / well. Equal amounts of serum were collected from each subject and pooled by group. The serum pool was serially diluted 2.5-fold in antibody dilution buffer containing Tween 20 and pneumococcal cell wall polysaccharide (CWPS) (5 μg / mL) obtained from Statens Serum Institute, followed by incubation at room temperature for 30 minutes. The plate was washed five times with wash buffer, and then 50 μl of pre-absorbed, 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 similarly, 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 as substrate to each well, followed by incubation at room temperature for 2 hours. The reaction was quenched by adding 50 μl of 3 M 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 capsular polysaccharides of serotypes 3 and 5 were conjugated to TT, the serotype-specific IgG concentrations were significantly higher than those of CRM 197 Rabbits immunized with PCV21(3 / 5 / 15B / 22F)-TT also demonstrated significantly elevated IgG concentrations against eight additional serotypes not present in PREVNAR13 (i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F). In particular, serotype 9N had a greater than 50-fold increase in serum-specific IgG concentrations compared to PREVNAR13.

[0184] Functional immunogenicity assay (MOPA)

[0185] Antibody function was assessed by testing serum samples in the MOPA assay. S. pneumoniae MOPA strains stored at -70°C or below were diluted to the corresponding final dilution factor to achieve a concentration of approximately 50,000 CFU / mL for each strain. Equal volumes of serum were collected from each subject, pooled by group, and serially diluted two-fold so that 20 μl of serum remained in a U-bottom plate. After dilution, 10 μl of the bacterial strain prepared for each serotype was mixed with the diluted sample. The mixture was incubated at room temperature for 30 minutes to ensure thorough mixing of S. pneumoniae and antibody. A mixture of predifferentiated HL-60 cells and complement was added, and the mixture was incubated for 45 minutes in a CO2 incubator (37°C). The temperature was lowered to stop phagocytosis, and 10 μl of the reaction solution was spotted onto agar plates that had been pre-dried for 30–60 minutes. The plates were then allowed to absorb for 20 minutes until dry. A 25 mg / mL TTC stock solution was added to the prepared overlay agar, followed by the appropriate antibody for the corresponding strain. The mixture was thoroughly mixed, and then approximately 25 mL of the mixture was added to the plate and allowed to harden for approximately 30 minutes. The fully hardened plates were incubated in a CO2 incubator (37°C) for 12 to 18 hours, after which colonies were counted. The MOPA titer was expressed as the dilution 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.

[0186] [Table 4]

[0187] When serotypes 3 and 5 were conjugated to TT, functional MOPA titers were higher than those of CRM 197 The MOPA titer was significantly increased compared to that obtained when the virus was complexed with PCV21( Rabbits immunized with (3 / 5 / 15B / 22F)-TT also demonstrated significantly elevated 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 immunogenicity titers were measured in the same manner as in 4-1, and the results of two separate experiments are shown below.

[0190] Serotype-specific IgG concentration measurement

[0191] [Table 5] TIFF2025128199000006.tif63170

[0192] When capsular polysaccharides of serotypes 1 and 5 were conjugated to TT, the serotype-specific IgG concentrations were significantly higher than those of CRM 197 Rabbits immunized with PCV21(1 / 5 / 15B / 22F)-TT also demonstrated significantly elevated 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 elevation (>50-fold) relative to PREVNAR13.

[0193] Functional immunogenicity assay (MOPA)

[0194] [Table 6]

[0195] When serotypes 1 and 5 were conjugated to TT, functional MOPA titers were higher than those of CRM 197Rabbits immunized with PCV21(3 / 5 / 15B / 22F)-TT also demonstrated significantly elevated functional MOPA titers against each of eight additional serotypes not present in PREVNAR13 (i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F).

[0196] Example 5. Additional details on the preparation of glycoprotein conjugates from Streptococcus pneumoniae serotype 9N

[0197] Preparation of cell banks

[0198] Streptococcus pneumoniae serotype 9N (ATCC 6309) was obtained from the American Type Culture Collection (ATCC). Seed stocks were cultured for several generations to propagate the strain and remove animal-derived components. Stock vials were kept in a refrigerator (<-70°C) with synthetic glycerol as a cryoprotectant. For the preparation of cell banks, cell cultures were grown in soy-based medium. Prior to freezing, cells were concentrated by centrifugation, and after removing the spent medium, the cell pellet was transferred to fresh medium containing a cryoprotectant (e.g., synthetic glycerol). and resuspended in

[0199] fermentation

[0200] Cultures from the cell bank were inoculated into seed bottles containing soy-based medium. The cultures were incubated at a constant temperature without agitation until growth conditions were met. The seed bottles were used to inoculate seed fermenters containing soy-based medium with controlled temperature, pH, and agitation rate. Fermentation was terminated after growth had stopped or after the fermenter's operating capacity was reached. Fermentation was terminated by adding an inactivating agent, followed by removal of cell debris using a combination of continuous-flow centrifugation and filtration.

[0201] purification

[0202] The purification process of 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 the calculated amount of WFI. If necessary, the reaction pH was adjusted to approximately 6.0. After pH adjustment, the reaction temperature was adjusted to 21–25°C. Approximately 0.024–0.189 mg of sodium periodate per mg of sugar was added to initiate oxidation. 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 on 10 times the diafiltration volume of WFI. The purified activated polysaccharide was then stored at 2-8°C. The purified activated polysaccharide was characterized by (i) glycoconjugate 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 weight of polysaccharides and polysaccharide-protein complexes. SEC is used to separate polysaccharides based on hydrodynamic volume. A refractive index (RI) detector and a multi-angle laser light scattering (MALLS) detector are used to determine molecular weight. When light interacts with a substance, it is scattered. The amount of scattered light is related to the concentration, the square of dn / dc (specific refractive index increment), and the molar mass of the material. The molecular weight is calculated based on the scattered light signal from the MALLS detector and the concentration signal from the RI detector.

[0207] The degree of oxidation (DO) of activated polysaccharides is determined as the moles of sugar repeat units divided by the moles of aldehyde, where the moles of sugar repeat units are determined using various colorimetric techniques, e.g., the anthrone assay, and the moles of aldehyde are determined by the Park-Johnson colorimetric assay.

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

[0209] complex

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

[0211] Activated polysaccharides and CRM 197 The lyophilized mixture was reconstituted in 0.1 M sodium phosphate solution and then thoroughly mixed. The final polysaccharide concentration in the reaction solution was approximately 10–20 g / L. Conjugation was initiated by adding 1.0–1.2 molar equivalents of sodium cyanoborohydride (NaBH3CN) to the reaction mixture, and the reaction was carried out at 35–39°C for 44–52 hours. The conjugation reaction was terminated by adding an equal volume of 0.9% sodium chloride solution to the conjugation reaction solution, followed by the addition of 1.8–2.2 molar equivalents of sodium borohydride (NaBH4) to cap any unreacted aldehydes. The capping reaction was carried out at 21–25°C for 3–6 hours.

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

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

[0214] The changes in the characteristics of the serotype 9N complex were observed with varying degrees of oxidation (DO), and the results are summarized in Table 7.

[0215] [Table 7]

[0216] Changes in the characteristics of the serotype 9N complex, activated polysaccharides, and CRM during freeze-drying 197 The results are summarized in Table 8.

[0217] [Table 8]

[0218] The changes in the characteristics of the serotype 9N conjugate were observed with varying polysaccharide concentrations in the conjugation reaction solution, and the results are summarized in Table 9.

[0219] [Table 9]

[0220] Example 6. Immunogenicity analysis

[0221] CRM 197 A monovalent conjugate composition was formulated containing a Streptococcus pneumoniae serotype 9 N glycobody-protein conjugate conjugated to

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

[0223] Five female New Zealand White rabbits weighing 2.5-3.5 kg were immunized intramuscularly at week 0 with the proposed human clinical dose (2.2 μg of conjugate; plus 0.25 mg / mL aluminum as AlPO4). The rabbits were re-immunized with the same dose of conjugate vaccine at week 2, and blood samples were collected at week 4. Serotype-specific ELISA was performed on serum samples at weeks 0 and 4.

[0224] The analytical results are shown in Table 10. Rabbits immunized with the monovalent conjugate composition (Conjugate No. 8) showed a significant increase in total IgG titers of serotype 9N. Rabbits immunized with the other conjugates also showed a significant increase in total IgG titers.

[0225] Table 10 shows the results of measuring IgG concentrations after immunizing rabbits with conjugate No. 8 from Table 8.

[0226] [Table 10]

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

[0228] The following references are cited in this application and provide general information about the art and provide assay and other details discussed in this application. The following references are incorporated herein by reference in their entirety.

[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]WHO 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. 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 conjugated to a capsular polysaccharide from a different serotype of Streptococcus pneumoniae, wherein the Streptococcus pneumoniae serotypes are 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 is a CRM １９７ or tetanus toxoid, Four of the capsular polysaccharides are conjugated to tetanus toxoid, and the remaining capsular polysaccharide is conjugated to CRM. １９７ and the four capsular polysaccharides conjugated to tetanus toxoid are serotypes 15B, 22F, and two serotypes selected from the group consisting of serotypes 1, 3, and 5.

2. The capsular polysaccharides from serotypes 1, 5, 15B, and 22F are conjugated to the 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 10. The mixed carrier multivalent pneumococcal conjugate composition of claim 1, wherein the pneumococcal conjugate composition is complexed with

3. The capsular polysaccharides from serotypes 1, 3, 15B, and 22F are conjugated to the tetanus toxoid, 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 10. The mixed carrier multivalent pneumococcal conjugate composition of claim 1, wherein the pneumococcal conjugate composition is complexed with

4. The capsular polysaccharides from serotypes 3, 5, 15B, and 22F are conjugated to the tetanus toxoid, and the capsular polysaccharides 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 10. The mixed carrier multivalent pneumococcal conjugate composition of claim 1, wherein the pneumococcal conjugate composition is complexed with

5. 10. The mixed carrier multivalent pneumococcal conjugate composition of any one of the preceding claims, further comprising an adjuvant.

6. 6. The mixed carrier multivalent pneumococcal conjugate composition of claim 5, wherein the adjuvant is an aluminum-based adjuvant.

7. 7. The mixed carrier multivalent pneumococcal conjugate composition of claim 6, wherein the adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide.

8. 8. The mixed carrier polyvalent pneumococcal conjugate composition of claim 7, wherein the adjuvant is aluminum phosphate.

9. 10. The mixed carrier multivalent pneumococcal conjugate composition of any one of the preceding claims, further comprising an adjuvant. The capsular polysaccharide from serotype 9N is activated so that the capsular polysaccharide 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 It is complexed with The capsular polysaccharide and CRM from serotype 9N １９７ The complex formed between has a molecular weight of 500 to 4,000 kDa, The capsular polysaccharide and CRM from serotype 9N １９７ CRM of the capsular polysaccharide from serotype 9N in the complex formed between １９７ is between 0.5 and 2.5 (w / w), and / or The capsular polysaccharide and CRM from serotype 9N １９７ 15-60% of the complex formed between ｄ 10. The mixed carrier multivalent pneumococcal conjugate composition of any one of the preceding claims, comprising:

10. 10. Use of the mixed carrier multivalent pneumococcal conjugate composition of any of the preceding claims for the prophylaxis against Streptococcus pneumoniae infection or disease in a subject.

11. A vaccine comprising the mixed carrier multivalent pneumococcal conjugate composition of any one of claims 1 to 9 and a pharmaceutically acceptable excipient.

12. 12. A method for preventing Streptococcus pneumoniae infection or disease in a subject, comprising administering to the subject a prophylactically effective amount of the mixed carrier multivalent pneumococcal conjugate composition of any one of claims 1 to 9 or the vaccine of claim 11.

13. 13. The method of claim 12, wherein the subject is a human at least 50 years of age and the disease is pneumonia or invasive pneumococcal disease (IPD).

14. 13. The method of claim 12, wherein 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).

15. 15. The method of claim 14, wherein the subject is between 6 weeks and 5 years old, between 2 and 15 months old, or between 6 and 17 years old.

16. The use according to claim 10 or the method according to any one of claims 12 to 15, wherein the subject is a human.

17. The method of any one of claims 12 to 16, wherein the mixed carrier multivalent pneumococcal conjugate composition or the vaccine is administered by intramuscular injection.

18. The method of any one of claims 12 to 17, wherein the mixed carrier multivalent pneumococcal conjugate composition or the vaccine is administered as part of an immunization series.

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