Multivalent pneumococcal polysaccharide-protein conjugate composition, and method of using the same

Abstract

To provide an improved multivalent pneumococcal conjugate composition and a vaccine containing the composition.SOLUTION: A multivalent pneumococcal conjugate composition contains 22 to 27 different kinds of pneumococcal capsular polysaccharide-protein conjugates, wherein each pneumococcal capsular polysaccharide-protein conjugate includes a protein carrier conjugated into capsular polysaccharide derived from a different serotype of streptococcus pneumoniae, and a streptococcus pneumoniae serotype is selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and 35B.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 / 949,164, filed December 17, 2019, and Korean Patent Application No. 10-2019-0093276, filed July 31, 2019, the entire disclosures of which are incorporated herein by reference.

[0002] Technical Field FIELD OF THE INVENTION This application relates generally to multivalent pneumococcal conjugate compositions, vaccines comprising the same, and methods of using such compositions and vaccines to prevent pneumococcal infection and disease in a subject. [Background technology]

[0003] Streptococcus pneumoniae (pneumococcus) is a lance-shaped, Gram-positive, facultative anaerobic bacterium with over 90 known serotypes. Most S. pneumoniae serotypes cause disease (such as pneumonia, bacteremia, meningitis, and otitis), and the 23 most common serotypes have been shown to account for approximately 90% of invasive disease worldwide. Serotypes are classified based on the serological response to capsular polysaccharides, the most important virulence factor for S. pneumoniae. Capsular polysaccharides are T-cell-independent antigens that induce antibody production even in the absence of helper T cells. T-cell-independent antigens generally induce low-affinity antibodies and elicit short-lived immune responses that leave little to no immunological memory.

[0004] Early pneumococcal vaccines contained combinations of capsular polysaccharides from different serotypes. These vaccines could confer immunity against Streptococcus pneumoniae in patients with developed or healthy immune systems, but were ineffective in children with underdeveloped immune systems and elderly subjects, whose immune functions are often weakened. To improve the immune response to pneumococcal vaccines, particularly in children and elderly subjects at higher risk of Streptococcus pneumoniae infection, capsular polysaccharides were conjugated to appropriate carrier proteins to produce pneumococcal conjugate vaccines. Conjugation to an appropriate carrier protein converts capsular polysaccharides from T-cell-independent antigens to T-cell-dependent antigens. Therefore, the immune response to the conjugated capsular polysaccharides involves helper T cells, thereby helping to induce stronger and more rapid immune responses upon re-exposure to the capsular polysaccharides.

[0005] There are at least two approaches to developing pneumococcal glycoconjugate 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 is an example of a single-carrier approach, and uses CRM, a non-toxic variant of diphtheria toxoid with a single amino acid substitution of glycine with glutamic acid. 197 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 most prevalent Streptococcus pneumoniae serotypes at the time of its license: 4, 6B, 9V, 14, 18C, 19F, and 23F. The 13-valent vaccine, PREVNAR13, contains the CRM 197 Serotypes 1, 5, 7F, 3, 6A, and 19A were added to the protein carrier. Merck has identified 13 serotypes present in PREVNAR13. In addition, 22F and 33F are CRM 197Merck is developing a 15-valent V114 vaccine, including one conjugated to CRM 197 Also disclosed is a 21-valent pneumococcal conjugate composition (PCV21) comprising at least one of the following 21 Streptococcus pneumoniae serotypes: 3, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 33F, 35B, and 15B, 15C, or de-O-acetylated 15B conjugated to a pneumococcal virus (see U.S. Patent No. 5,929,499).

[0006] A second pneumococcal conjugate vaccine approach is the mixed carrier approach. In this 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 using Haemophilus influenzae protein D, tetanus toxoid, and diphtheria toxoid as protein carriers. In SYNFLORIX, serotypes 1, 4, 5, 6B, 7F, 9V, 14, and 23F are conjugated to protein D; serotype 18C is conjugated to tetanus toxoid; and serotype 19F is conjugated to diphtheria toxoid. (Non-Patent Document 1) More recently, Sanofi Pasteur and SK Biosciences have developed 16-valent (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F, and 33F), 20-valent (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 13F, 14F, 15F, 16F, 17F, 18F, 19F, 22F, 23F, and 33F), 20-valent (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 19F, 23F, 24F, 25F, 26F, 27F, 28F, 29F, 30F, 31F, 32F, 33F), 34F, 35F, 36F, 37F, 38F, 39F, 40F, 41F, 42F, 43F, 44F, 45F, 46F, 47F, 48F, 49F, 50F, 5 The authors have produced 21-valent (1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F) and 21-valent (1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F) mixed-carrier pneumococcal conjugate vaccines, each of which is incorporated by reference in its entirety. In these mixed-carrier multivalent pneumococcal conjugate vaccines, two serotypes (two of serotypes 1, 3, and 5) or four serotypes (serotypes 15B and 22F and two of serotypes 1, 3, and 5) are conjugated to tetanus toxoid, with the remaining serotypes being CRM. 197 It is conjugated to

[0007] Both single-carrier and mixed-carrier glycoconjugate vaccines have been used to provide varying levels of protection against pneumococcal serotypes included in the vaccine, however, serotype substitution, or an increased prevalence of pathogenic pneumococcal strains / serotypes not included in the glycoconjugate vaccine, has been observed and is a concern. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 8,192,746 [Patent Document 2] US2019 / 0192648 [Patent Document 3] WO2018 / 027123 [Patent Document 4] WO2018 / 027126 [Patent Document 5] WO2019 / 152921 [Patent Document 6] WO2019 / 152925 [Non-patent literature]

[0009] [Non-Patent Document 1] Vesikari et al., PIDJ, 28(4):S66~76(2009) [Non-patent document 2] Daniels et al., J Pediatr Pharmacol Ther.2016 Jan-Feb;21(1):27~35 Summary of the Invention [Means for solving the problem]

[0010] The present application provides new and improved multivalent pneumococcal conjugate compositions and vaccines comprising the same. In one aspect, the present application provides a multivalent pneumococcal conjugate composition comprising 22 to 27 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, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B. Other relevant Streptococcus pneumoniae serotypes may also be added to the multivalent pneumococcal conjugate composition. In certain embodiments, each capsular polysaccharide is conjugated to the same protein carrier. In certain embodiments, referred to as hybrid carrier embodiments, more than one type of protein carrier is used, e.g., two different protein carriers. For example, in certain embodiments, a particular capsular polysaccharide is conjugated to a first protein carrier, and the remaining capsular polysaccharide is attached to a second protein carrier. In certain embodiments, the first and second protein carriers are conjugated to a CRM. 197 and tetanus toxoid. In certain embodiments, two of the capsular polysaccharides are conjugated to tetanus toxoid, and the remaining capsular polysaccharide is CRM 197 In certain embodiments, the two capsular polysaccharides conjugated to the tetanus toxoid are selected from the group consisting of serotypes 1, 3, and 5. In certain embodiments, the two capsular polysaccharides conjugated to the tetanus toxoid are selected from the group consisting of serotypes 1, 3, 5, 15B, and 22F. In certain embodiments, four of the capsular polysaccharides are conjugated to the tetanus toxoid, with the remaining capsular polysaccharide being CRM 197In certain embodiments, four capsular polysaccharides are conjugated to tetanus toxoid, and two of the four capsular polysaccharides conjugated to the 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.

[0011] In one aspect, the multivalent pneumococcal conjugate composition comprises 27 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, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B.

[0012] In certain embodiments, capsular polysaccharides from serotypes 1 and 5 are conjugated to tetanus toxoid, and capsular polysaccharides from serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B are conjugated to CRM. 197 In another embodiment, capsular polysaccharides from serotypes 1 and 3 are conjugated to tetanus toxoid, and capsular polysaccharides from serotypes 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B are conjugated to CRM. 197 In yet another embodiment, capsular polysaccharides from serotypes 3 and 5 are conjugated to tetanus toxoid, and capsular polysaccharides from serotypes 1, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, Capsular polysaccharides derived from 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B are CRMs.197 It is conjugated to

[0013] In certain embodiments, 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.

[0014] In one embodiment, the mixed carrier multivalent pneumococcal conjugate composition comprises 27 different pneumococcal capsular polysaccharide-protein conjugates, with capsular polysaccharides from serotypes 1, 5, 15B, and 22F conjugated to tetanus toxoid, and capsular polysaccharides from serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 23A, 23B, 23F, 24F, 33F, and 35B conjugated to CRM. 197 It is conjugated to

[0015] In another embodiment, the mixed carrier multivalent pneumococcal conjugate composition comprises 27 different pneumococcal capsular polysaccharide-protein conjugates, wherein 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, 15A, 15C, 18C, 19A, 19F, 23A, 23B, 23F, 24F, 33F, and 35B are conjugated to CRM. 197 It is conjugated to

[0016] In another embodiment, the mixed carrier multivalent pneumococcal conjugate composition comprises 27 different pneumococcal capsular polysaccharide-protein conjugates, wherein 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, 15A, 15C, 18C, 19A, 19F, 23A, 23B, 23F, 24F, 33F, and 35B are conjugated to CRM. 197 It is conjugated to

[0017] In certain embodiments, the multivalent pneumococcal conjugate composition comprises 26 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, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B.

[0018] In certain embodiments, the multivalent pneumococcal conjugate composition comprises 25 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, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B.

[0019] In certain embodiments, the multivalent pneumococcal conjugate composition comprises 24 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, 8A, 8B, 9A, 9B, 10A, 10B, 11A, 11C, 12A, 12C, 13A, 13B, 14A, 14B, 15A, 15B, 16A, 16B, 17A, 17C, 18A, 18B, 19A, 20A, 21A, 22A, 23A, 24A, 25A, 26A, 27A, 28A, 29A, 30A, 31A, 32A, 33A, 34A, 35A, 36A, 37A, 38A, 39A, 40A, 41A, 42A, 43A, 44A, 45A, 46A, 47A, 48A, 49A, 50A, 51A, 52A, 53A, 54A, 55A, 56A, 57A, 58A, 59A, 60A, 61A, 62A, 63A, 64A, 65A, 66A, 67A, 68A, 69A, 70A, 71A, 72A, , 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B.

[0020] In certain embodiments, the multivalent pneumococcal conjugate composition comprises 23 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, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B.

[0021] In certain embodiments, the multivalent pneumococcal conjugate composition comprises 22 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, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B.

[0022] In some embodiments, the 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.

[0023] Another aspect is directed to the use of the multivalent pneumococcal conjugate composition as a vaccine.

[0024] Yet another aspect is directed to a vaccine comprising a multivalent pneumococcal conjugate composition and a pharmaceutically acceptable excipient.

[0025] Yet another aspect is directed to a method of preventing pneumococcal infection or disease in a subject, such as a human, comprising administering to the subject a prophylactically effective amount of a multivalent pneumococcal conjugate composition or a vaccine comprising the same.

[0026] 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).

[0027] In other embodiments, the subject is a human who is 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.

[0028] In certain embodiments, the multivalent pneumococcal conjugate composition or vaccine is administered by intramuscular injection. In certain embodiments, the multivalent pneumococcal conjugate composition or vaccine is administered as part of an immunization series.

[0029] Yet another aspect is directed to immunogenic compositions and methods for preparing the same, comprising at least one polysaccharide-protein conjugate, wherein the polysaccharide in the at least one polysaccharide-protein conjugate is a capsular polysaccharide from Streptococcus pneumoniae serotype 15A.

[0030] Yet another aspect is directed to immunogenic compositions and methods for preparing the same, comprising at least one polysaccharide-protein conjugate, wherein the polysaccharide in the at least one polysaccharide-protein conjugate is a capsular polysaccharide from Streptococcus pneumoniae serotype 15C.

[0031] Yet another aspect is directed to immunogenic compositions and methods for preparing the same, comprising at least one polysaccharide-protein conjugate, wherein the polysaccharide in the at least one polysaccharide-protein conjugate is a capsular polysaccharide from Streptococcus pneumoniae serotype 23A.

[0032] Yet another aspect is directed to immunogenic compositions and methods for preparing the same, comprising at least one polysaccharide-protein conjugate, wherein the polysaccharide in the at least one polysaccharide-protein conjugate is a capsular polysaccharide from Streptococcus pneumoniae serotype 23B.

[0033] Yet another aspect is directed to immunogenic compositions and methods for preparing the same, comprising at least one polysaccharide-protein conjugate, wherein the polysaccharide in the at least one polysaccharide-protein conjugate is a capsular polysaccharide from Streptococcus pneumoniae serotype 24F.

[0034] Yet another aspect is directed to immunogenic compositions and methods for preparing the same, comprising at least one polysaccharide-protein conjugate, wherein the polysaccharide in the at least one polysaccharide-protein conjugate is a capsular polysaccharide from Streptococcus pneumoniae serotype 35B.

[0035] The foregoing and other objects, features, and advantages of the pneumococcal conjugate composition will become more apparent from the following detailed description.

[0036] definition In order that this disclosure may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be found throughout the specification.

[0037] 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 described herein and / or which will become apparent to those skilled in the art upon reading this disclosure, and so forth.

[0038] 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 several routes, for example, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal, intradermal, etc.

[0039] Approximately: As used herein, the term "approximately" or "about" as applied to one or more applicable values ​​refers to a value that is close to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a value that is closer to the stated reference value in either direction (greater or less), unless otherwise specified or clear from the context. Refers to a range of values ​​that fall within 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or a lower percentage (unless such number exceeds 100% of all possible values).

[0040] Conjugate: As used herein and understood from the appropriate context, the term "conjugate" or "glycoconjugate" refers to a S. pneumoniae polysaccharide conjugated to a carrier protein using any covalent or non-covalent bioconjugation strategy.

[0041] Degree of oxidation: As used herein, the term "degree of oxidation" (DO) refers to the number of sugar repeat units per aldehyde group produced when purified or sized sugars are activated with an oxidizing agent. The degree of oxidation of sugars can be determined using routine methods known to those skilled in the art.

[0042] Embodiment: As used in this document, the terms “in one particular embodiment,” “in some embodiments,” and the like refer to embodiments of all aspects of the present disclosure, unless the context clearly dictates otherwise.

[0043] Excipient: As used herein, the term "excipient" refers to a non-therapeutic agent that can be included in a composition to, for example, obtain or contribute to a desired consistency or stabilizing effect.

[0044] Mixed Carrier: As used herein, mixed carrier pneumococcal conjugate composition refers to a pneumococcal conjugate composition having two or more types of protein carriers.

[0045] 22-valent pneumococcal conjugate composition: As used herein, the term "22-valent pneumococcal conjugate composition" or "PCV-22" refers to a composition comprising pneumococcal capsular polysaccharide-protein conjugates that comprises or consists of 22 different pneumococcal capsular polysaccharide-protein conjugates, wherein each pneumococcal capsular polysaccharide-protein conjugate comprises a protein carrier conjugated to a capsular polysaccharide from a different serotype of Streptococcus pneumoniae, and wherein the Streptococcus pneumoniae serotypes are selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B.

[0046] 23-valent pneumococcal conjugate composition: As used herein, the term "23-valent pneumococcal conjugate composition" or "PCV-23" refers to a composition comprising pneumococcal capsular polysaccharide-protein conjugates that comprises or consists of 23 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, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B.

[0047] 24-valent pneumococcal conjugate composition: As used herein, the term "24-valent pneumococcal conjugate composition" or "PCV-24" refers to a composition containing pneumococcal capsular polysaccharide-protein conjugates that comprises or consists of 24 different pneumococcal capsular polysaccharide-protein conjugates, each pneumococcal capsular polysaccharide-protein conjugate being a protein conjugated to a capsular polysaccharide from a different serotype of Streptococcus pneumoniae. The antibody comprises a protein carrier, and the Streptococcus pneumoniae serotype is selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B.

[0048] 25-valent pneumococcal conjugate composition: As used herein, the term "25-valent pneumococcal conjugate composition" or "PCV-25" refers to a composition comprising pneumococcal capsular polysaccharide-protein conjugates that comprises or consists of 25 different pneumococcal capsular polysaccharide-protein conjugates, wherein each pneumococcal capsular polysaccharide-protein conjugate comprises a protein carrier conjugated to a capsular polysaccharide from a different serotype of Streptococcus pneumoniae, and wherein the Streptococcus pneumoniae serotypes are selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B.

[0049] 26-valent pneumococcal conjugate composition: As used herein, the term "26-valent pneumococcal conjugate composition" or "PCV-26" refers to a composition comprising pneumococcal capsular polysaccharide-protein conjugates that comprises or consists of 26 different pneumococcal capsular polysaccharide-protein conjugates, wherein each pneumococcal capsular polysaccharide-protein conjugate comprises a protein carrier conjugated to a capsular polysaccharide from a different serotype of Streptococcus pneumoniae, and wherein the Streptococcus pneumoniae serotypes are selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B.

[0050] 27-valent pneumococcal conjugate composition: As used herein, the term "27-valent pneumococcal conjugate composition" or "PCV-27" refers to a composition comprising pneumococcal capsular polysaccharide-protein conjugates that comprises or consists of 27 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 pneumococcal serotypes being 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B.

[0051] Molecular weight: Unless otherwise specified, as used in this document, the term "molecular weight" of a capsular saccharide or capsular saccharide-carrier protein conjugate refers to the average molecular weight calculated by size exclusion chromatography (SEC) coupled with multi-angle laser light scattering (MALLS).

[0052] Multivalent: As used in this document, the term "multivalent" refers to a pneumococcal conjugate composition having pneumococcal capsular polysaccharides from two or more Streptococcus pneumoniae serotypes.

[0053] Pharmaceutically acceptable excipients: Pharmaceutically acceptable excipients that can be used in the present disclosure are conventional. Remington's Pharmaceutical Sciences by E.W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975) describes compositions and formulations suitable for the pharmaceutical delivery of one or more therapeutic compositions, such as vaccines, and additional pharmaceutical agents. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, propylene glycol, propylene glycol stearate ... Examples of suitable excipients include ethanol, glycols, water, ethanol, and the like. Generally, the type of excipient will depend on the particular mode of administration being used. For example, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, buffered 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, pH buffering agents, and the like, such as sodium acetate or sorbitan monolaurate.

[0054] Prophylactically effective amount: As defined in this document, the term "prophylactically effective amount" or "prophylactically effective dose" refers to the amount or dose required to elicit an immune response sufficient to delay the onset and / or reduce the frequency and / or severity of one or more symptoms caused by Streptococcus pneumoniae infection.

[0055] Prevention: As used herein, the term "prevention" refers to avoiding the onset of, delaying the onset of, and / or reducing the frequency and / or severity of one or more symptoms of a particular disease, disorder, or condition (e.g., Streptococcus pneumoniae infection). In some embodiments, an agent is considered to provide prevention for a particular disease, disorder, or condition if assessment of prevention on a population basis shows a statistically significant reduction in the occurrence, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition in a population susceptible to the particular disease, disorder, or condition.

[0056] 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 child. In some embodiments, the terms "individual" or "patient" are used and are intended to be interchangeable with "subject." DETAILED DESCRIPTION OF THE INVENTION

[0057] The following description of the disclosed embodiments and examples is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses.

[0058] The present application provides new and improved multivalent pneumococcal conjugate compositions and vaccines comprising the same. As shown in the Examples, robust antibody responses were observed against 27 serotypes in PCV-27, including serotypes not covered by existing pneumococcal vaccines, such as serotypes 15A, 15C, 23A, 23B, 24F, and 35B.

[0059] Pneumococcus polysaccharide serotype 15A Serotype 15A polysaccharides can be obtained directly from 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. Additionally, synthetic protocols can be used to produce 15A oligosaccharides.

[0060] Serotype 15A S. pneumoniae strains can be obtained from established cell culture collections (eg, the Streptococcal Reference Laboratory at the Centers for Disease Control and Prevention, Atlanta, GA) or from clinical specimens.

[0061] Bacterial cells are typically grown in media such as soy-based media. After fermentation by the membrane polysaccharide-producing bacterial cells, the bacterial cells are lysed to produce a cell lysate from which the serotype 15A polysaccharide can then be isolated using purification techniques known in the art, including centrifugation, depth filtration, precipitation, ultrafiltration, activated carbon treatment, diafiltration, and / or column chromatography (including, but not limited to, the methods disclosed in U.S. Patent Application Publication No. 2006 / 0228380).

[0062] The purified serotype 15A polysaccharide is conjugated to a carrier protein to form an immunogenic composition comprising at least one polysaccharide-protein conjugate comprising serotype 15A polysaccharide and a carrier protein. In one embodiment, the 15A polysaccharide-protein conjugate is (i) subjecting purified Streptococcus pneumoniae serotype 15A polysaccharide to an acid hydrolysis reaction and heat or a microfluidizer, and then reacting with an oxidizing agent to produce activated Streptococcus pneumoniae serotype 15A polysaccharide; (ii) optionally, lyophilizing the activated S. pneumoniae serotype 15A polysaccharide and carrier protein; (iii) suspending the activated S. pneumoniae serotype 15A polysaccharide and carrier protein in dimethyl sulfoxide (DMSO); (iv) reacting the activated S. pneumoniae serotype 15A polysaccharide and the carrier protein with a reducing agent to produce a S. pneumoniae serotype 15A polysaccharide-carrier protein conjugate; and (v) capping any unreacted aldehydes in the Streptococcus pneumoniae serotype 15A polysaccharide-carrier protein conjugate to produce an immunogenic conjugate comprising Streptococcus pneumoniae serotype 15A polysaccharide covalently linked to a carrier protein. Further details regarding the reagents (e.g., oxidizing agents, reducing agents, carrier proteins, etc.) and conditions that can be used in this method are disclosed elsewhere in this application, including in subsequent sections and examples.

[0063] Activated serotype 15A capsular polysaccharide can be characterized by various parameters, including, for example, molecular weight (MW) and / or degree of oxidation (Do).

[0064] In one aspect, the activated Streptococcus pneumoniae serotype 15A polysaccharide has a molecular weight of less than 120 kDa prior to conjugation, including, for example, activated serotype 15A capsular polysaccharides having a molecular weight of about 10-120 kDa, 50-120 kDa, 70-120 kDa, 70-80 kDa, 70-118 kDa, 114-118 kDa, or about 116 kDa prior to conjugation. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0065] In one aspect, when the molecular weight of the S. pneumoniae serotype 15A polysaccharide is less than 120 kDa prior to conjugation, polysaccharide-protein conjugates of about 1,000 to 5,000 kDa can be produced, e.g., about 1,200 to 4,000 kDa, 1,200 to 1,500 kDa, 1,200 to 3,500 kDa, 1,400 to 4,000 kDa, about 1,200 kDa, about 1,400 kDa, or about 4,000 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0066] The purified serotype 15A polysaccharide may also be characterized by its degree of oxidation after activation with an oxidizing agent. In one aspect, the activated serotype 15A polysaccharide may have a degree of oxidation ranging from 1 to 15, such as 4 to 10, 4 to 8, 4 to 5, 5 to 8, or about 4.

[0067] In one aspect, activated polysaccharide of Streptococcus pneumoniae serotype 15A having an oxidation level (Do) of about 4 is conjugated to a carrier protein to obtain a serotype 15A capsular polysaccharide-protein conjugate having a free polysaccharide (free PS) content of 40% or less, e.g., 5-40%, 20-40%, 25-40%, 20-35%, 25-35%, 30-35%.

[0068] Polysaccharides may undergo slight size reduction during normal purification procedures. In addition, as described in this disclosure, polysaccharides may be subjected to sizing before conjugation. The molecular weight ranges referred to above refer to the molecular weight ranges of purified polysaccharides after a final sizing step (e.g., after purification, hydrolysis, and activation) before conjugation.

[0069] Pneumococcus polysaccharide serotype 15C Serotype 15C polysaccharides can be obtained directly from 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. Additionally, synthetic protocols can be used to produce 15C oligosaccharides.

[0070] Serotype 15C S. pneumoniae strains can be obtained from established cell culture collections (e.g., the Streptococcal Reference Laboratory at the Centers for Disease Control and Prevention, Atlanta, Georgia) or from clinical specimens. Alternatively, serotype 15C polysaccharide can be obtained by de-O-acetylation of serotype 15B polysaccharide, usually by alkali treatment.

[0071] Bacterial cells are typically grown in a medium such as a soy-based medium. After fermentation by the bacterial cells that produce Streptococcus pneumoniae serotype 15C capsular polysaccharide, the bacterial cells are lysed to produce a cell lysate. The serotype 15C polysaccharide can then be isolated from the cell lysate using purification techniques known in the art, including centrifugation, depth filtration, precipitation, ultrafiltration, activated carbon treatment, diafiltration, and / or column chromatography (including, but not limited to, the methods disclosed in U.S. Patent Application Publication No. 2006 / 0228380).

[0072] The purified serotype 15C polysaccharide is conjugated to a carrier protein to form an immunogenic composition comprising at least one polysaccharide-protein conjugate comprising serotype 15C polysaccharide and a carrier protein. In one embodiment, the 15C polysaccharide-protein conjugate is (i) reacting purified Streptococcus pneumoniae serotype 15C polysaccharide with an oxidizing agent to produce activated Streptococcus pneumoniae serotype 15C polysaccharide; (ii) optionally, lyophilizing the activated S. pneumoniae serotype 15C polysaccharide and carrier protein; (iii) suspending the activated S. pneumoniae serotype 15C polysaccharide and carrier protein in dimethyl sulfoxide (DMSO) or phosphate buffer; (iv) reacting the mixture of activated serotype 15C polysaccharide and carrier protein with a reducing agent to produce a serotype 15C polysaccharide-carrier protein conjugate; and (v) capping any unreacted aldehydes in the serotype 15C polysaccharide-carrier protein conjugate to produce an immunogenic conjugate comprising Streptococcus pneumoniae serotype 15C polysaccharide covalently linked to a carrier protein. Further details regarding the reagents (e.g., oxidizing agents, reducing agents, carrier proteins, etc.) and conditions that can be used in this method are provided in the following sections: are disclosed elsewhere in this application, including in subsequent sections and examples.

[0073] Activated serotype 15C capsular polysaccharide can be characterized by various parameters, including, for example, molecular weight (MW) and / or degree of oxidation (Do).

[0074] In one aspect, the activated S. pneumoniae serotype 15C polysaccharide prior to conjugation may have a molecular weight of 200-1,000 kDa, such as 400-800 kDa, 500-775 kDa, 470-775 kDa, 500-770 kDa, 520-680 kDa, 510-770 kDa, 510-550 kDa, 670-770 kDa, or a similar molecular weight range. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0075] 15C polysaccharide-protein conjugates having molecular weights of about 1,000 to 10,000 kDa can be produced, for example, about 2,000 to 6,000 kDa, 2,500 to 5,000 kDa, 6,000 to 10,000 kDa, or 6,200 to 9,400 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0076] The purified serotype 15C polysaccharide may also be characterized by its degree of oxidation after activation with an oxidizing agent. In one embodiment, the activated serotype 15C polysaccharide may have a degree of oxidation ranging from 1 to 40. By adding sodium periodate to S. pneumoniae serotype 15C polysaccharide, a degree of oxidation of 8 to 35, 15 to 35, 8 to 20, 8 to 9, 9 to 20, or 30 to 35 can be achieved.

[0077] In one aspect, activated polysaccharide of Streptococcus pneumoniae serotype 15C having an oxidation level (Do) of 30-35 is conjugated to a carrier protein to obtain a serotype 15C capsular polysaccharide-protein conjugate having a free polysaccharide (free PS) content of 40% or less, e.g., 5-40%, 20-40%, 25-40%, 20-35%, 25-35%, or 30-35%.

[0078] Polysaccharides may undergo slight size reduction during normal purification procedures. In addition, as described in this disclosure, polysaccharides may be subjected to sizing before conjugation. The molecular weight ranges referred to above refer to the molecular weight ranges of purified polysaccharides after a final sizing step (e.g., after purification, hydrolysis, and activation) before conjugation.

[0079] Pneumococcus polysaccharide serotype 23A Serotype 23A polysaccharides can be obtained directly from 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. Additionally, synthetic protocols can be used to produce 23A oligosaccharides.

[0080] Serotype 23A S. pneumoniae strains can be obtained from established cell culture collections (eg, the Streptococcal Reference Laboratory at the Centers for Disease Control and Prevention, Atlanta, GA) or from clinical specimens.

[0081] Bacterial cells are typically grown in a medium such as a soy-based medium. After fermentation with bacterial cells producing S. pneumoniae serotype 23A capsular polysaccharide, the bacterial cells are lysed to produce a cell lysate. This can then be purified by centrifugation, depth filtration, sedimentation, ultrafiltration, activated carbon treatment, diafiltration, or other methods. Serotype 23A polysaccharide can be isolated from the cell lysate using purification techniques known in the art, including but not limited to, methods disclosed in U.S. Patent Application Publication No. 2006 / 0228380, including but not limited to, filtration and / or column chromatography.

[0082] The purified serotype 23A polysaccharide is conjugated to a carrier protein to form an immunogenic composition comprising at least one polysaccharide-protein conjugate comprising serotype 23A polysaccharide and a carrier protein. In one embodiment, the 23A polysaccharide-protein conjugate is (i) reacting purified Streptococcus pneumoniae serotype 23A with an oxidizing agent to produce activated Streptococcus pneumoniae serotype 23A polysaccharide; (ii) optionally, lyophilizing the activated S. pneumoniae serotype 23A polysaccharide and carrier protein; (iii) suspending the activated S. pneumoniae serotype 23A polysaccharide and carrier protein in dimethyl sulfoxide (DMSO) or phosphate buffer; (iv) reacting the mixture of activated S. pneumoniae serotype 23A polysaccharide and carrier protein with a reducing agent to produce a S. pneumoniae serotype 23A polysaccharide-carrier protein conjugate; and (v) capping any unreacted aldehydes in the Streptococcus pneumoniae serotype 23A polysaccharide-carrier protein conjugate to produce an immunogenic conjugate comprising Streptococcus pneumoniae serotype 23A polysaccharide covalently linked to a carrier protein. Further details regarding the reagents (e.g., oxidizing agents, reducing agents, carrier proteins, etc.) and conditions that can be used in this method are disclosed elsewhere in this application, including in subsequent sections and examples.

[0083] Activated serotype 23A capsular polysaccharide can be characterized by various parameters including, for example, molecular weight (MW) and / or degree of oxidation (Do).

[0084] In one aspect, the activated S. pneumoniae serotype 23A polysaccharide prior to conjugation may have a molecular weight of 300-700 kDa, such as 400-650 kDa, 430-650 kDa, 470-650 kDa, 470-570 kDa, 470-490 kDa, or similar molecular weight ranges. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0085] Using the methods disclosed herein, serotype 23A polysaccharide-protein conjugates of approximately 2,000-7,000 kDa can be produced. The molecular weight of the serotype 23A capsular polysaccharide-protein conjugate can be about 2,000-4,000 kDa, 4,000-7,000 kDa, 4,200-6,700 kDa, 4,350-6,650 kDa, 5,000-6,700 kDa, about 4,300 kDa, about 5,000 kDa, or about 6,600 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0086] Purified serotype 23A polysaccharide may also be characterized by its degree of oxidation after activation with an oxidizing agent. In one aspect, activated serotype 23A polysaccharide may have a degree of oxidation ranging from 4 to 25, such as 6 to 24, 6 to 18, 9 to 18, 6 to 9, 6 to 10, 6 to 11, or 9 to 11.

[0087] In one embodiment, an activated polysaccharide of Streptococcus pneumoniae serotype 23A having an oxidation level (Do) of 9-11 is conjugated to a carrier protein to provide an activated polysaccharide of 40% or less, for example, 5-40%, 20-40%, 25-40%, 20-35%, 25-35%, or 30-35%. A serotype 23A capsular polysaccharide-protein conjugate is obtained having a free polysaccharide (free PS) content of 100%.

[0088] Any suitable buffer solution may be used for conjugation, including DMSO or phosphate buffer. When DMSO is used, the reaction concentration of the polysaccharide can be 2.5 mg / mL or less, including, for example, 1.0 mg / mL to 2.5 mg / mL, 1.0 mg / mL to 2.0 mg / mL, or 1.0 mg / mL to 1.5 mg / mL. When phosphate buffer is used, the reaction concentration of the polysaccharide can be 10 to 20 mg / mL, including, for example, 15 mg / mL.

[0089] Polysaccharides may undergo slight size reduction during normal purification procedures. In addition, as described in this disclosure, polysaccharides may be subjected to sizing prior to conjugation.

[0090] Pneumococcus polysaccharide serotype 23B Serotype 23B polysaccharides can be obtained directly from 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. Additionally, synthetic protocols can be used to produce 23B oligosaccharides.

[0091] Serotype 23B S. pneumoniae strains can be obtained from established cell culture collections (eg, the Streptococcal Reference Laboratory at the Centers for Disease Control and Prevention, Atlanta, GA) or from clinical specimens.

[0092] Bacterial cells are typically grown in a medium such as a soy-based medium. After fermentation by the bacterial cells that produce Streptococcus pneumoniae serotype 23B capsular polysaccharide, the bacterial cells are lysed to produce a cell lysate. The serotype 23B polysaccharide can then be isolated from the cell lysate using purification techniques known in the art, including centrifugation, depth filtration, precipitation, ultrafiltration, activated carbon treatment, diafiltration, and / or column chromatography (including, but not limited to, the methods disclosed in U.S. Patent Application Publication No. 2006 / 0228380).

[0093] The purified serotype 23B polysaccharide is conjugated to a carrier protein to form an immunogenic composition comprising at least one polysaccharide-protein conjugate comprising serotype 23B polysaccharide and a carrier protein. In one embodiment, the 23B polysaccharide-protein conjugate is (i) reacting purified Streptococcus pneumoniae serotype 23B with an oxidizing agent to produce activated Streptococcus pneumoniae serotype 23B polysaccharide; (ii) optionally, lyophilizing the activated S. pneumoniae serotype 23B polysaccharide and carrier protein; (iii) suspending the activated S. pneumoniae serotype 23B polysaccharide and carrier protein in dimethyl sulfoxide (DMSO); (iv) reacting the mixture of activated S. pneumoniae serotype 23B polysaccharide and carrier protein with a reducing agent to produce a S. pneumoniae serotype 23B polysaccharide-carrier protein conjugate; and (v) capping any unreacted aldehydes in the Streptococcus pneumoniae serotype 23B polysaccharide-carrier protein conjugate to produce an immunogenic conjugate comprising Streptococcus pneumoniae serotype 23B polysaccharide covalently linked to a carrier protein. Reagents that can be used in this method Further details regarding the reagents (eg, oxidizing agents, reducing agents, carrier proteins, etc.) and conditions are disclosed elsewhere in this application, including in subsequent sections and examples.

[0094] Activated serotype 23B capsular polysaccharide can be characterized by various parameters including, for example, molecular weight (MW) and / or degree of oxidation (Do).

[0095] In one aspect, the activated Streptococcus pneumoniae serotype 23B polysaccharide prior to conjugation may have a molecular weight of 100-800 kDa, such as 200-700 kDa, 200-650 kDa, 300-650 kDa, 380-640 kDa, 550-675 kDa, 200-250 kDa, 220-230 kDa, 220-225 kDa, or a similar molecular weight range. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0096] The methods disclosed herein can be used to produce serotype 23B polysaccharide-protein conjugates of approximately 2,000-7,000 kDa. The molecular weight of the serotype 23B capsular polysaccharide-protein conjugate can range from approximately 2,000-4,000 kDa, 2,000-5,000, 4,000-7,000 kDa, 2,400-6,800 kDa, 4,600-6,800 kDa, or 6,400-6,800 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0097] The purified serotype 23B polysaccharide may also be characterized by its degree of oxidation after activation with an oxidizing agent. In one embodiment, the activated serotype 23B polysaccharide may have an oxidation degree of 5.4 or less, e.g., an oxidation degree of 1 to 5.4, 2 to 5.4, 2.3 to 5.4, 2 to 3, or 2.3 to 2.8.

[0098] In one aspect, activated polysaccharide of Streptococcus pneumoniae serotype 23B (as discussed above) having an oxidation level (Do) of 3 or less is conjugated to a carrier protein to obtain a serotype 23B capsular polysaccharide-protein conjugate having a free polysaccharide (free PS) content of 40% or less, e.g., 5-40%, 20-40%, 25-40%, 20-35%, 25-35%, or 30-35%.

[0099] Polysaccharides may undergo slight size reduction during normal purification procedures. In addition, as described in this disclosure, polysaccharides may be subjected to sizing prior to conjugation.

[0100] Pneumococcus polysaccharide serotype 24F Serotype 24F polysaccharides can be obtained directly from 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. Additionally, synthetic protocols can be used to produce 24F oligosaccharides.

[0101] Serotype 24F S. pneumoniae strains can be obtained from established cell culture collections (eg, the Streptococcal Reference Laboratory at the Centers for Disease Control and Prevention, Atlanta, GA) or from clinical specimens.

[0102] Bacterial cells are typically grown in a medium such as a soy-based medium. After fermentation by the bacterial cells producing S. pneumoniae serotype 24F capsular polysaccharide, the bacterial cells are lysed to produce a cell lysate. The cell lysate can then be purified by any method known in the art, including centrifugation, depth filtration, sedimentation, ultrafiltration, activated carbon treatment, diafiltration, and / or column chromatography. Serotype 24F polysaccharide can be isolated from cell lysates using various purification techniques, including but not limited to, the methods disclosed in US Patent Application Publication No. 2006 / 0228380.

[0103] The purified serotype 24F polysaccharide is conjugated to a carrier protein to form an immunogenic composition comprising at least one polysaccharide-protein conjugate comprising serotype 24F polysaccharide and a carrier protein. In one embodiment, the 24F polysaccharide-protein conjugate is (i) subjecting purified Streptococcus pneumoniae serotype 24F polysaccharide to an acid hydrolysis reaction or a microfluidizer, followed by reaction with an oxidizing agent to produce activated Streptococcus pneumoniae serotype 24F polysaccharide; (ii) optionally, lyophilizing the activated S. pneumoniae serotype 24F polysaccharide and the carrier protein; (iii) suspending the activated S. pneumoniae serotype 24F polysaccharide and carrier protein in dimethyl sulfoxide (DMSO) or phosphate buffer; (iv) reacting the activated S. pneumoniae serotype 24F polysaccharide and the carrier protein with a reducing agent to produce a S. pneumoniae serotype 24F polysaccharide-carrier protein conjugate; and (v) capping any unreacted aldehydes in the Streptococcus pneumoniae serotype 24F polysaccharide-carrier protein conjugate to produce an immunogenic conjugate comprising Streptococcus pneumoniae serotype 24F polysaccharide covalently linked to a carrier protein. Further details regarding the reagents (e.g., oxidizing agents, reducing agents, carrier proteins, etc.) and conditions that can be used in this method are disclosed elsewhere in this application, including in subsequent sections and examples.

[0104] Activated serotype 24F capsular polysaccharide can be characterized by various parameters including, for example, molecular weight (MW) and / or degree of oxidation (Do).

[0105] In one aspect, the activated S. pneumoniae serotype 24F polysaccharide prior to conjugation may have a molecular weight of 100-500 kDa, such as 150-350 kDa, 200-400 kDa, 200-300 kDa, 225-275 kDa, 240-260 kDa, 245-255 kDa, about 250 kDa, or a similar molecular weight range. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0106] The methods disclosed herein can be used to produce serotype 24F polysaccharide-protein conjugates of approximately 1,000-5,000 kDa. The molecular weight of the serotype 24F capsular polysaccharide-protein conjugates can range from approximately 1,500-5,000 kDa, 2,000-4,500, or 2,500-3,500 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0107] Purified serotype 24F polysaccharide may also be characterized by its degree of oxidation after activation with an oxidizing agent. In one embodiment, activated serotype 24F polysaccharide may have a degree of oxidation of at least 90, including about 90-100.

[0108] In one embodiment, in the step of reacting activated serotype 24F polysaccharide having an oxidation degree of at least 90 with a carrier protein, 2.0 or less molar equivalents of a reducing agent can be used to obtain a serotype 24F capsular polysaccharide-protein conjugate having 40% or less, e.g., 5-40%, 20-40%, 25-40%, 20-35%, 25-35%, or 30-35% free saccharide (free PS). 0.5-1.2, 1.0-1.2, or about 1.2 molar equivalents of a reducing agent may be used.

[0109] Polysaccharides may undergo slight size reduction during normal purification procedures. In addition, as described in this disclosure, polysaccharides may be subjected to sizing prior to conjugation.

[0110] Pneumococcus polysaccharide serotype 35B Serotype 35B polysaccharides can be obtained directly from 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. Additionally, synthetic protocols can be used to produce the 35B oligosaccharides.

[0111] Serotype 35B S. pneumoniae strains can be obtained from established cell culture collections (eg, the Streptococcal Reference Laboratory at the Centers for Disease Control and Prevention, Atlanta, GA) or from clinical specimens.

[0112] Bacterial cells are typically grown in a medium such as a soy-based medium. After fermentation by the bacterial cells that produce Streptococcus pneumoniae serotype 35B capsular polysaccharide, the bacterial cells are lysed to produce a cell lysate. The serotype 35B polysaccharide can then be isolated from the cell lysate using purification techniques known in the art, including centrifugation, depth filtration, precipitation, ultrafiltration, activated carbon treatment, diafiltration, and / or column chromatography (including, but not limited to, the methods disclosed in U.S. Patent Application Publication No. 2006 / 0228380).

[0113] The purified serotype 35B polysaccharide is conjugated to a carrier protein to form an immunogenic composition comprising at least one polysaccharide-protein conjugate comprising serotype 35B polysaccharide and a carrier protein. In one embodiment, the 35B polysaccharide-protein conjugate is (i) reacting purified S. pneumoniae serotype 35B with an oxidizing agent to produce activated S. pneumoniae serotype 35B polysaccharide; (ii) optionally, lyophilizing the activated S. pneumoniae serotype 35B polysaccharide and carrier protein; (iii) suspending the activated S. pneumoniae serotype 35B polysaccharide and carrier protein in dimethyl sulfoxide (DMSO) or phosphate buffer; (iv) reacting the activated S. pneumoniae serotype 35B polysaccharide and the carrier protein with a reducing agent to produce a S. pneumoniae serotype 35B polysaccharide-carrier protein conjugate; and (v) capping any unreacted aldehydes in the Streptococcus pneumoniae serotype 35B polysaccharide-carrier protein conjugate to produce an immunogenic conjugate comprising Streptococcus pneumoniae serotype 35B polysaccharide covalently linked to a carrier protein. Further details regarding the reagents (e.g., oxidizing agents, reducing agents, carrier proteins, etc.) and conditions that can be used in this method are disclosed elsewhere in this application, including in subsequent sections and examples.

[0114] Activated serotype 35B capsular polysaccharide can be characterized by various parameters including, for example, molecular weight (MW) and / or degree of oxidation (Do).

[0115] For example, purified serotype 35B polysaccharide can be reduced in size by, for example, high pressure homogenization or mechanical homogenization prior to conjugation to a carrier protein. In one embodiment, activated serotype 35B polysaccharide can be reduced in size by, for example, high pressure homogenization or mechanical homogenization prior to conjugation. The molecular weight of the antibody is 10 to 20,000 kDa, 10 to 1,000 kDa, 10 to 500 kDa, 10 to 300 kDa, 20 to 200 kDa, or 20 to 120 kDa.

[0116] Purified serotype 35B polysaccharide can be characterized by its degree of oxidation (Do) after activation with an oxidizing agent. In one aspect, activated serotype 35B polysaccharide can have a Do of 1-50, 1-45, 1-40, 1-35, 1-30, 2-50, 2-45, 2-40, 2-35, 2-30, 3-40, 3-35, 3-30, 4-40, 4-35, or 4-30.

[0117] In one aspect, activated polysaccharide of Streptococcus pneumoniae serotype 35B having an oxidation level (Do) of 4 to 30 is conjugated to a carrier protein to obtain a serotype 35B capsular polysaccharide-protein conjugate having a free polysaccharide (free PS) content of 40% or less, for example, 5-40%, 20-40%, 25-40%, 20-35%, 25-35% or 30-35%.

[0118] To generate serotype 35B glycoconjugates with advantageous immunogenic properties, one or more of the following process parameters in the activation (oxidation), conjugation and / or capping steps can be combined: In the activation step, periodic acid (e.g., sodium or potassium periodate) is reacted with 0.005-0.5, 0.005-0.3, 0.005-0.2, or 0.007-0.15 molar equivalents per mole of serotype 35B polysaccharide; The activation step can be carried out in an aqueous solvent such as sodium acetate buffer or deionized water. The activation step can be carried out in a 0.1 mM to 15 mM or 0.1 to 10 mM sodium acetate buffer; The activation step can be carried out at pH 4-8 or pH 4-7.5. In the activation step, periodic acid can be treated at 21°C to 25°C. In the activation step, periodic acid and serotype 35B polysaccharide can be reacted for 0.5 to 50 hours or 1 to 25 hours; After the activation step, the activated serotype 35B polysaccharide can be concentrated, for example, using a 30 kDa MWCO ultrafiltration filter; In the conjugation step, the concentration of the activated serotype 35B polysaccharide in the conjugation reaction may be 5 mg / mL to 30 mg / mL or 10 mg / mL to 20 mg / mL; In the conjugation step, the initial loading ratio of carrier protein and activated serotype 35B polysaccharide (PR:PS) may be 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9 or 1:3, preferably 1:0.5-2; In the conjugation step, the amount of reducing agent used may be 0.1 to 5 moles or 0.5 to 2 equivalents per mole of activated polysaccharide, preferably 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mole equivalents per mole of activated polysaccharide, or more preferably 0.8 to 1.6 mole equivalents of reducing agent per mole of activated polysaccharide; In the conjugation step, the temperature may be 20°C to 45°C, 30°C to 40°C, 35°C to 40°C, or 37±2°C; In the conjugation step, the pH may be 5.5 to 8.5, 5.5 to 7.5, or 6 to 7.5; In the conjugation step, the carrier protein and the activated serotype 35B polysaccharide can be reacted with the reducing agent for 1 to 70 hours or 40 to 60 hours; after the conjugation step, the yield of serotype 35B glycoconjugate may be at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%; In the capping step, sodium borohydride can be used in an amount of 0.5 to 5 molar equivalents per mole of activated serotype 35B polysaccharide, such as 1 to 3 or 1.5 to 2.5 molar equivalents per mole of activated serotype 35B polysaccharide, or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 molar equivalents of sodium borohydride per mole of activated polysaccharide, In the capping step, the temperature may be 10 to 40°C, 15 to 30°C, 20 to 26°C, or 23±2°C; In the capping step, the reaction time may be 0.5 to 10 hours or 2 to 8 hours; and / or After the capping step, the serotype 35B glycoconjugate is added to, for example, a 100 kDa Concentration can be achieved using a MWCO ultrafiltration filter.

[0119] In one exemplary embodiment, the method for producing a serotype 35B glycoconjugate comprises the following steps: (i) diluting the isolated serotype 35B polysaccharide with sodium acetate buffer (NaOAc, pH 4.5 to pH 6.0) or deionized water (DW); (ii) reacting serotype 35B polysaccharide with 0.005 to 0.5 molar equivalents of sodium periodate to produce activated serotype 35B polysaccharide; (iii) purifying the activated serotype 35B polysaccharide and then mixing it with a cryoprotectant; (iv) lyophilizing the activated serotype 35B and the carrier protein, respectively; (v) resuspending the activated serotype 35B polysaccharide and carrier protein in DMSO or phosphate buffer; (vi) mixing the resuspended activated serotype 35B polysaccharide with a carrier protein and reacting with sodium cyanoborohydride to produce a serotype 35B polysaccharide-carrier protein conjugate; (vii) capping any unreacted aldehydes in the serotype 35B polysaccharide-carrier protein conjugate with sodium borohydride; and (viii) obtaining an immunogenic conjugate comprising Streptococcus pneumoniae serotype 35B polysaccharide covalently linked to a carrier protein Includes.

[0120] In another exemplary embodiment, the method for producing a serotype 35B glycoconjugate comprises the steps of: (i) diluting the isolated serotype 35B polysaccharide with sodium acetate buffer (NaOAc, pH 4.5 to pH 6.0) or deionized water (DW); (ii) reacting serotype 35B polysaccharide with 0.005 to 0.5 molar equivalents of sodium periodate to produce activated serotype 35B polysaccharide; (iii) purifying the activated serotype 35B polysaccharide; (iv) mixing the activated serotype 35B polysaccharide with a carrier protein followed by co-lyophilization; (v) simultaneously resuspending the lyophilized activated serotype 35B polysaccharide and carrier protein in DMSO or phosphate buffer; (vi) Serotype 35B polysaccharide-carrier protein by reaction with sodium cyanoborohydride producing a protein conjugate; (vii) capping any unreacted aldehydes in the serotype 35B polysaccharide-carrier protein conjugate with sodium borohydride; and (viii) obtaining an immunogenic conjugate comprising Streptococcus pneumoniae serotype 35B polysaccharide covalently linked to a carrier protein Includes.

[0121] Pneumococcus polysaccharide serotype 22F Activated serotype 22F capsular polysaccharide can be characterized by various parameters, including, for example, the degree of oxidation (Do) after activation with an oxidizing agent. In certain embodiments, activated serotype 22F polysaccharide may have a Do of 20-100, 20-80, 20-60, 20-50, 20-40, 20-35, 25-100, 25-50, 25-35, 28-32, or 29-31. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0122] Serotype 22F polysaccharide-protein conjugates can be characterized by various parameters, including, for example, the protein-to-polysaccharide (PS / PR) ratio after conjugation, free sugars (free PS), MSD % or molecular weight (MALLS). In certain embodiments, the PS / PR ratio of a 22F capsular polysaccharide-protein conjugate (e.g., 22F-TT) may be 0.2-1.5, 0.2-0.5, 0.3-0.4, 0.6-1.0, 0.7-0.9, or 0.6-0.8. In certain embodiments, a 22F capsular polysaccharide-protein conjugate (e.g., 22F-TT) has 40% or less free PS, e.g., 2-40%, 2-20%, 2-10%, 5-30%, 10-25%, 15-25%, 17-21%, or about 19%. In certain embodiments, a serotype 22F capsular polysaccharide-protein conjugate (e.g., 22F-TT) has an MSD (%) of 5-60%, 5-10%, 5-50%, 10-50%, 25-50%, 40-50%, 42-46%, or about 44%. In certain embodiments, the molecular weight of a 22F capsular polysaccharide-protein conjugate (e.g., 22F-TT) may be in the range of about 1,000-6,000 kDa, 2,000-5,000 kDa, 2,500-4,000 kDa, 3,000-3,500 kDa, or 3,000-3,100 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0123] Any of the above parameters for serotype 22F can be combined as described. For example, in certain embodiments, the activated serotype 22F polysaccharide used to generate the serotype 22F polysaccharide-protein conjugate has a Do of about 29-31 and a protein (TT) to polysaccharide reactivity ratio of about 1:1. In certain embodiments, the final conjugate has a polysaccharide / carrier protein ratio (PS / PR) of about 0.6-0.8, free PS of about 17-21%, MSD% of about 42-46%, and optionally a molecular weight by MALLS of about 3,000-3,100 kDa.

[0124] Pneumococcus polysaccharide serotype 15B Activated serotype 15B capsular polysaccharide can be characterized by various parameters, including, for example, the degree of oxidation (Do) after activation with an oxidizing agent. In certain embodiments, activated serotype 15B polysaccharide may have a degree of oxidation of 1 to 15, 5 to 10, 6 to 8, or about 7. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0125] Serotype 15B polysaccharide-protein conjugates can be characterized, for example, by the protein to polysaccharide (PS / PR) ratio after conjugation, free sugar (free PS), MSD % or molecular weight (M The 15B capsular polysaccharide-protein conjugates (e.g., 15B-TT) can be characterized by various parameters, including free PS / PR (PS / PR ratio). In certain embodiments, the PS / PR ratio of the 15B capsular polysaccharide-protein conjugates (e.g., 15B-TT) can be 0.2-1.5, 0.2-0.5, 0.3-0.4, 0.6-1.0, 0.7-0.9, or 0.8-1.0. In certain embodiments, the 15B capsular polysaccharide-protein conjugates (e.g., 15B-TT) have 30% or less, e.g., 2-30%, 2-20%, 2-10%, 5-10%, 8-10%, or about 9% free PS. In certain embodiments, a serotype 15B capsular polysaccharide-protein conjugate (e.g., 15B-TT) has an MSD (%) of 50-90%, 60-85%, 65-80%, 70-80%, 74-78%, or about 76%. In certain embodiments, the molecular weight of a 15B capsular polysaccharide-protein conjugate (e.g., 15B-TT) may be in the range of about 2,000-15,000 kDa, 10,000-15,000 kDa, 2,000-10,000 kDa, 3,000-7,500 kDa, 4,000-6,000 kDa, 5,000-6,000 kDa, or 5,500-5,600 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0126] Any of the above parameters for serotype 15B can be combined as described. For example, in certain embodiments, the activated serotype 15B polysaccharide used to generate the serotype 15B polysaccharide-protein conjugate has a Do of about 7.0 and a protein (TT) to polysaccharide reactivity ratio of about 1.25:1. In certain embodiments, the final conjugate has a polysaccharide / carrier protein ratio (PS / PR) of about 0.8-1.0, free PS of about 8-10%, MSD% of about 74-78%, and optionally a molecular weight by MALLS of about 5,500-5,600.

[0127] Pneumococcus polysaccharide serotype 19A Activated serotype 19A capsular polysaccharide can be characterized by various parameters, including, for example, the degree of oxidation (Do) after activation with an oxidizing agent. In certain embodiments, activated serotype 19A polysaccharide may have a Do of 20-40, 30-40, 35-40, 30-35, 20-30, 22-28, 24-28, 25-30, or 25-27. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0128] Serotype 19A polysaccharide-protein conjugates can be characterized by various parameters, including, for example, the protein to polysaccharide (PS / PR) ratio after conjugation, the free sugars (free PS), the MSD (%), or the molecular weight (MALLS). In certain embodiments, 19A capsular polysaccharide-protein conjugates (e.g., 19A-CRM 197 ) may have a PS / PR ratio of 0.2 to 1.5, 0.2 to 0.5, 0.3 to 0.4, 0.6 to 1.0, 0.7 to 0.9, or 0.6 to 0.8. In certain embodiments, the 19A capsular polysaccharide-protein conjugate (e.g., 19A-CRM 197) has 50% or less, e.g., 10-40%, 15-40%, 20-40%, 25-40%, 25-35%, 30-40%, 30-35%, 32-34%, or about 33% free PS. In certain embodiments, serotype 19A capsular polysaccharide-protein conjugates (e.g., 19A-CRM 197 ) has an MSD (%) of 35-70%, 40-50%, 50-70%, 60-70%, 63-68%, or about 65%. In certain embodiments, serotype 19A capsular polysaccharide-protein conjugates (e.g., 19A-CRM 197 The molecular weight of ) may range from about 2,000 to 8,000 kDa, 3,500 to 7,000 kDa, 4,500 to 6,500 kDa, 5,000 to 6,500 kDa, or 5,250 to 6,250 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0129] Any of the above parameters for serotype 19A can be combined as described. For example, in certain embodiments, the activated serotype 19A polysaccharide used to make the serotype 19A polysaccharide-protein conjugate has a Do of about 25-27 and a protein to protein ratio of about 1:1. 197 In certain embodiments, the final conjugate has a polysaccharide / carrier protein ratio (PS / PR) of about 0.7, a free PS of about 30-35%, an MSD% of about 63-68%, and optionally a molecular weight by MALLS of about 5,250-6,250.

[0130] Pneumococcus polysaccharide serotype 19F Activated serotype 19F capsular polysaccharide can be characterized by various parameters, including, for example, the degree of oxidation (Do) after activation with an oxidizing agent. In certain embodiments, activated serotype 19F polysaccharide may have a Do of 20-50, 30-50, 40-50, 25-35, 20-30, 22-28, 25-30, 23-27, or 24-26. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0131] Serotype 19F polysaccharide-protein conjugates can be characterized by various parameters, including, for example, the protein to polysaccharide (PS / PR) ratio after conjugation, MSD (%), or free saccharide (free PS). In certain embodiments, 19F capsular polysaccharide-protein conjugates (e.g., 19F-CRM 197 ) may have a PS / PR ratio of 0.2 to 1.5, 0.2 to 0.5, 0.3 to 0.4, 0.6 to 1.0, 0.7 to 0.9, or 0.6 to 0.8. In certain embodiments, serotype 19F capsular polysaccharide-protein conjugates (e.g., 19F-CRM 197 ) has an MSD (%) of 25-80%, 35-75%, 40-60%, 70-80%, 75-80%, or about 77%. In certain embodiments, serotype 19F capsular polysaccharide-protein conjugates (e.g., 19F-CRM 197 ) has 30% or less, for example, 2-30%, 2-20%, 2-10%, 2-9%, 3-7%, 4-6%, or about 5% free PS.

[0132] Any of the above parameters of serotype 19F can be combined as described. For example, in certain embodiments, the activated serotype 19F polysaccharide used to make the serotype 19F polysaccharide-protein conjugate has a Do of about 24-26 and a protein to protein (CRM) ratio of about 1.5:1. 197 In certain embodiments, the polysaccharide / carrier protein ratio (PS / PR) in the final conjugate is about 0.7, the free PS is about 4-6%, and the MSD% is about 75-80%.

[0133] Pneumococcus polysaccharide serotype 4 Serotype 4 polysaccharide-protein conjugates can be characterized by various parameters, including, for example, the protein to polysaccharide (PS / PR) ratio after conjugation, the free sugars (free PS), the MSD (%), or the molecular weight (MALLS). In certain embodiments, 4 capsular polysaccharide-protein conjugates (e.g., 4-CRM 197 ) may have a PS / PR ratio of 0.2 to 1.5, 0.8 to 1.1, 0.8 to 1.3, 0.9 to 1.1, or about 1.0. In certain embodiments, the serotype 4 capsular polysaccharide-protein conjugate (e.g., 4-CRM 197 ) has 40% or less, e.g., 5-30%, 15-35%, 5-15%, 7-13%, 9-11%, or about 10% free PS. In certain embodiments, serotype 4 capsular polysaccharide-protein conjugates (e.g., 4-CRM 197 ) has an MSD (%) of 40-80%, 45-75%, 45-55%, 60-75%, or 70-75%. In certain embodiments, a serotype 4 capsular polysaccharide-protein conjugate (e.g., 4-CRM 197 ) molecular weight can be in the range of about 500 to 2,500 kDa, 500 to 1,000 kDa, 1,000 to 2,000 kDa, 1,500 to 2,000 kDa, 1,800 to 2,000 kDa, or 1,850 to 1,950 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0134] Any of the above parameters for serotype 4 can be combined as described. For example, in certain embodiments, the activated serotype 4 polysaccharide used to make the serotype 4 polysaccharide-protein conjugate has a Do of about 1.4 and a protein (CRM) ratio of about 1.25:1. 197 In certain embodiments, the final conjugate has a polysaccharide / carrier protein ratio (PS / PR) of about 1.0, a free PS of about 9-11%, an MSD% of about 70-75%, and optionally a molecular weight by MALLS of about 1,850-1,950.

[0135] Pneumococcus polysaccharide serotype 9V Serotype 9V polysaccharide-protein conjugates can be characterized by various parameters, including, for example, the protein to polysaccharide (PS / PR) ratio after conjugation, the free sugars (free PS), the MSD (%), or the molecular weight (MALLS). In certain embodiments, the 9V capsular polysaccharide-protein conjugates (e.g., 9V-CRM 197 ) may have a PS / PR ratio of 0.2 to 1.5, 0.2 to 0.5, 0.3 to 0.4, 0.8 to 1.3, 1.0 to 1.2, or about 1.1. In certain embodiments, serotype 9V capsular polysaccharide-protein conjugates (e.g., 9V-CRM 197 ) has 35% or less, e.g., 10-35%, 20-35%, 5-15%, 7-13%, 9-11%, or about 10% free PS. In certain embodiments, serotype 9V capsular polysaccharide-protein conjugates (e.g., 9V-CRM 197 ) has an MSD (%) of 40-80%, 45-75%, 45-60%, 50-65%, 55-65%, 57-61%, or about 59%. In certain embodiments, a serotype 9V capsular polysaccharide-protein conjugate (e.g., (9V-CRM 197 The molecular weight of the hydroxyl group may range from about 500 to 2,000 kDa, 500 to 1,500 kDa, 1,000 to 2,000 kDa, 1,000 to 1,500 kDa, 1,000 to 1,200 kDa, or 1,100 to 1,200 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0136] Any of the above parameters for serotype 9V can be combined as described. For example, in certain embodiments, the activated serotype 9V polysaccharide used to make the serotype 9V polysaccharide-protein conjugate has a Do of about 7.4 and a protein (CRM) ratio of about 1.25:1. 197In certain embodiments, the final conjugate has a polysaccharide / carrier protein ratio (PS / PR) of about 1.1, a free PS of about 9-11%, an MSD% of about 57-61%, and optionally a molecular weight by MALLS of about 1,100-1,200.

[0137] Multivalent pneumococcal conjugate compositions and methods for making same The present disclosure provides multivalent pneumococcal conjugate compositions comprising 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. Various aspects and embodiments of the multivalent pneumococcal conjugate compositions are described herein.

[0138] In one aspect, the multivalent pneumococcal conjugate composition comprises pneumococcal capsular polysaccharide-protein conjugates comprising or consisting of 22 to 27 different pneumococcal capsular polysaccharide-protein conjugates, each pneumococcal capsular polysaccharide-protein conjugate comprising: The protein carrier comprises a capsular polysaccharide from a different serotype of Streptococcus pneumoniae, wherein the Streptococcus pneumoniae serotype is selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B.

[0139] In one embodiment, a multivalent pneumococcal conjugate composition comprises pneumococcal capsular polysaccharide-protein conjugates that comprise or consist of 27 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 pneumococcal serotypes being 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B. This multivalent pneumococcal conjugate composition is also referred to as PCV-27.

[0140] In one aspect, a multivalent pneumococcal conjugate composition comprises pneumococcal capsular polysaccharide-protein conjugates that comprise or consist of 26 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, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B. This multivalent pneumococcal conjugate composition is also referred to as PCV-26. In certain embodiments of PCV-26, at least one of the Streptococcus pneumoniae serotypes is 35B. In certain embodiments of PCV-26, the Streptococcus pneumoniae serotypes comprise or consist of four serotypes selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and 35B, and 15A, 15C, 23A, 23B, and 24F. For example, PCV-26 comprises pneumococcal capsular polysaccharide-protein conjugates that may comprise or consist of 26 different pneumococcal capsular polysaccharide-protein conjugates, each comprising a protein carrier conjugated to a capsular polysaccharide from a different serotype of Streptococcus pneumoniae, the Streptococcus pneumoniae serotypes being: a) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15A, 15C, 23A, and 23B; b) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15A, 15C, 23A, and 24F; c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15A, 15C, 23B, and 24F; d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15A, 23A, 23B, and 24F; or e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15C, 23A, 23B, and 24F is.

[0141] In one embodiment, the multivalent pneumococcal conjugate composition comprises or consists of 25 different pneumococcal capsular polysaccharide-protein conjugates. The polyvalent pneumococcal conjugate composition comprises a protein carrier conjugated to a capsular polysaccharide from a different serotype of Streptococcus pneumoniae, wherein each pneumococcal capsular polysaccharide-protein conjugate comprises 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, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B. This multivalent pneumococcal conjugate composition is also referred to as PCV-25. In certain embodiments of PCV-25, at least one of the Streptococcus pneumoniae serotypes is 35B. In certain embodiments of PCV-25, the Streptococcus pneumoniae serotypes comprise or consist of three serotypes selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and 35B, and 15A, 15C, 23A, 23B, and 24F. For example, PCV-25 comprises pneumococcal capsular polysaccharide-protein conjugates that may comprise or consist of 25 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, and the Streptococcus pneumoniae serotypes are: a) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15A, 15C, and 23A; b) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15A, 15C, and 23B; c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15A, 15C, and 24F; d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15A, 23A, and 23B; e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15A, 23A, and 24F; f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15A, 23B, and 24F; g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15C, 23A, and 23B; h) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15C, 23A, and 24F; i) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15C, 23B, and 24F; or j)1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 23A, 23B, and 24F is.

[0142] In one aspect, the multivalent pneumococcal conjugate composition comprises pneumococcal capsular polysaccharide-protein conjugates that comprise or consist of 24 different pneumococcal capsular polysaccharide-protein conjugates, each pneumococcal capsular polysaccharide-protein conjugate comprising a different The polyvalent pneumococcal conjugate composition comprises a protein carrier conjugated to a capsular polysaccharide from a Streptococcus pneumoniae serotype selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B. This multivalent pneumococcal conjugate composition is also referred to as PCV-24. In certain embodiments of PCV-24, at least one of the Streptococcus pneumoniae serotypes is 35B. In certain embodiments of PCV-24, the Streptococcus pneumoniae serotypes comprise or consist of two serotypes selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and 35B, and 15A, 15C, 23A, 23B, and 24F. For example, PCV-24 comprises pneumococcal capsular polysaccharide-protein conjugates that may comprise or consist of 24 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, and the Streptococcus pneumoniae serotypes are: a) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15A, and 15C; b) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15A, and 23A; c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15A, and 23B; d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15A, and 24F; e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15C, and 23A; f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15C, and 23B; g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 15C, and 24F; h) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 23A, and 23B; i) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 23A, and 24F; or j) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, 23B, and 24F is.

[0143] In one aspect, the multivalent pneumococcal conjugate composition comprises pneumococcal capsular polysaccharide-protein conjugates that comprise or consist of 23 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 pneumococcal serotype is selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B. This multivalent pneumococcal conjugate composition is also referred to as PCV-23. In certain embodiments of PCV-23, at least one of the pneumococcal serotypes is 35B. In certain embodiments of PCV-23, the Streptococcus pneumoniae serotype comprises or consists of one serotype selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and 35B, and 15A, 15C, 23A, 23B, and 24F. For example, PCV-23 comprises pneumococcal capsular polysaccharide-protein conjugates that may comprise or consist of 23 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, and the Streptococcus pneumoniae serotypes are: a) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, and 15A; b) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, and 15C; c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, and 23A; d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, and 23B; or e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, 35B, and 24F is.

[0144] In one aspect, a multivalent pneumococcal conjugate composition comprises pneumococcal capsular polysaccharide-protein conjugates that comprise or consist of 22 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, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B. This multivalent pneumococcal conjugate composition is also referred to as PCV-22. For example, PCV-22 comprises pneumococcal capsular polysaccharide-protein conjugates that may comprise or consist of 22 different pneumococcal capsular polysaccharide-protein conjugates, each comprising a protein carrier conjugated to a capsular polysaccharide from a different serotype of Streptococcus pneumoniae, the Streptococcus pneumoniae serotypes being: a) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and 15A; b) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and 15C; c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and 23A; d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and 23B; e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and 24F; or f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and 35B is.

[0145] PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, and PCV-27 embodiments may include applicable Streptococcus pneumoniae serotypes other than serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B. For example, in certain embodiments, PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27 further includes one or more of Streptococcus pneumoniae serotypes 2, 12A, 16F, 17F, 20A, 20B, 20F, 31, 45, and 46. In certain embodiments, PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27 further comprises one or more of Streptococcus pneumoniae serotypes 6C, 6D, 7B, 7C, 18B, 21, 22A, 24B, 27, 28A, 34, 35F, 38, and 39. Other relevant Streptococcus pneumoniae serotypes may also be added to any one of PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27.

[0146] One or more of the Streptococcus pneumoniae serotypes PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27 may be replaced with one or more applicable Streptococcus pneumoniae serotypes other than serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B. For example, in certain embodiments, one or more of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B in PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27 are replaced with one or more of Streptococcus pneumoniae serotypes 2, 12A, 14, 16F, 20A, 20B, 20F, 31, 45, and 46. In certain embodiments, one or more of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B in PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27 are replaced with one or more of Streptococcus pneumoniae serotypes 6C, 6D, 7B, 7C, 18B, 21, 22A, 24B, 27, 28A, 34, 35F, 38, and 39. One or more of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B in any one of PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27 may be replaced with other applicable Streptococcus pneumoniae serotypes.

[0147] Carrier proteins In polysaccharide-protein conjugate vaccines, a carrier protein is conjugated to a polysaccharide antigen to form a glycoconjugate. The carrier protein serves to enhance the immune response (e.g., antibody response) to the polysaccharide antigen. The carrier protein must be suitable for conjugation with pneumococcal polysaccharides using standard conjugation procedures. It is a must.

[0148] Carrier proteins that can be used in glycoconjugates include, but are not limited to, DT (diphtheria toxoid), TT (tetanus toxoid), fragment C of TT, CRM 197(genetically induced non-toxic mutants of diphtheria toxin that retain the immunological properties of wild-type diphtheria toxin), other genetically induced diphtheria toxin variants (e.g., CRM176, CRM228, CRM45 (Uchida et al. (1973) J. Biol. Chem. 218:3838-3844), CRM9, CRM102, CRM103, or CRM107; and Nicholls and Youle in Genetically Engineered Toxins, Eds.: Frankel, Marcel Dekker Other mutations described by Inc (1992); deletion of Glu-148 or mutation to Asp, Gln, or Ser and / or deletion of Ala158 or mutation to Gly and other mutations disclosed in U.S. Pat. Nos. 4,709,017 and 4,950,740; mutation of at least one or more residues Lys516, Lys526, Phe530, and / or Lys534 and other mutations disclosed in U.S. Pat. Nos. 5,917,017 and 6,455,673; or fragments disclosed in U.S. Pat. No. 5,843,711, ply detoxified in some way, for example, pneumococcal pneumolysin (ply), including dPLY-GMBS (WO 2004 / 081515, WO 2006 / 032499) or dPLY-formol (Kuo et al. (1995) Infect Immun 63:2706-2713), PhtX including PhtA, PhtB, PhtD, PhtE (the sequences of PhtA, PhtB, PhtD or PhtE are disclosed in WO00 / 37105 and WO00 / 39299) and fusions of Pht proteins, for example, PhtDE fusions, PhtBE fusions, PhtAE (WO01 / 98334, WO03 / 054007, WO2009 / 000826), OMPC (meningococcal outer membrane protein) commonly extracted from Neisseria meningitidis serogroup B (EP0372501), PorB (N.meningitidis), PD (Haemophilus influenza protein D; see, for example, EP 0 594 610 B) or immunologically functional equivalents thereof, synthetic peptides (EP 0 378 881, EPO 427 347), heat shock proteins (WO 93 / 17712, WO 94 / 03208), pertussis proteins (WO 98 / 58668, EPO 471 177), cytokines, lymphokines, growth factors or hormones (WO 91 / 01146), N19 protein (Baraldoi et al. (2004) Infect. Immun 72:4884-4887), artificial proteins containing multiple human CD4+ T cell epitopes derived from antigens derived from various pathogens, such as the pneumococcal surface protein PspA (WO 02 / 091998), iron uptake protein (WO 01 / 72337), Clostridium difficile toxin A or B (WO 00 / 61761), transferrin-binding protein, pneumococcal adhesion protein (PsaA), and recombinant Pseudomonas aeruginosa exotoxin A, particularly its non-toxic mutants, such as exotoxin A with a substitution at glutamic acid 553 (Douglas et al. (1987) J. Bacteriol. 169(11):4967-4971) (Falugi et al. (2001) Eur J Immunol 31:3816-3824). Other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or purified protein derivative of tuberculin (PPD) can also be used as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins, such as cholera toxoid (e.g., as described in WO2004 / 083251), Escherichia coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa, immunologically functional equivalents of which can also be used as carrier proteins in the present invention. When each of the carrier proteins is referred to herein, it is understood that the immunologically functional equivalent is intended to be illustrative. It is understood to include.

[0149] In certain embodiments, the carrier protein of the glycoconjugate is TT (including fragment C of TT), DT (CRM 197 and others discussed above), PD, PhtX, PhtD, PhtDE fusions (particularly those disclosed in WO01 / 98334 and WO03 / 054007), detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, Clostridium difficile toxin A or B, and PsaA. When each carrier protein is mentioned herein, it is understood to encompass its immunologically functional equivalent. For example, those skilled in the art will understand that DT mutants that are immunologically functional equivalents, including but not limited to those discussed above, are also included when DT is mentioned herein.

[0150] In certain embodiments, the carrier protein of the glycoconjugate is DT (diphtheria toxoid), CRM 197 , TT (tetanus toxoid), fragment C of TT and PD (protein D of Haemophilus influenzae).

[0151] In one embodiment, the carrier protein of the glycoconjugate of the present invention may be DT (diphtheria toxoid). Naturally occurring or wild-type diphtheria toxin can be obtained from toxigenic strains available from various public sources, including the American Type Culture Collection (ATCC). As used herein, the term DT (diphtheria toxoid) is intended to include all DT variants that function as its functional equivalents. Such DT mutants include, but are not limited to, for example, CRM176, CRM228, CRM45, CRM9, CRM102, CRM103 or CRM107; a mutation or deletion of Glu148 to Asp compared to wild-type DT (disclosed in U.S. Pat. No. 4,709,017); a deletion or mutation of Glu148 to Asp, and a deletion or mutation of Ala158 to Gly, as disclosed in U.S. Pat. Nos. 4,709,017 and 4,950,740; a mutation of at least one or more residues selected from the group consisting of Lys516, Lys526, Phe530 and Lys534, as disclosed in U.S. Pat. No. 5,917,017, and a mutation of Glu148, Glu349, Lys516 and / or Phe530, as disclosed in U.S. Pat. No. 6,455,673; and in U.S. Pat. No. 5,843,711. In one embodiment the isolated capsular saccharide is a CRM 197 Conjugated to a protein. CRM 197 The protein is a non-toxic form of diphtheria toxin that retains the immunological properties of wild-type diphtheria toxin. 197 is a non-toxic phage β generated by nitrosoguanidine mutagenesis of toxigenic corynephage β. 197 CRM is produced by tox-infected Corynebacterium diphtheriae (Uchida et al. (1971) Nature New Biology 233:8-11). 197The protein has the same molecular weight as diphtheria toxin but differs by a single base change (guanine to adenine) in the structural gene. This single base change causes an amino acid substitution (glutamic acid for glycine) in the mature protein, eliminating the toxic properties of diphtheria toxin. CRM 197 Proteins are safe and effective T cell-dependent carriers of sugars. 197 and further details regarding its production can be found, for example, in US Pat. No. 5,614,382, which is incorporated herein by reference in its entirety.

[0152] In another embodiment, the carrier protein of the glycoconjugate is TT (tetanus toxoid). Tetanus toxoid is produced worldwide for mass immunization against tetanus (or trismus) caused by Clostridium tetani. Tetanus toxoid has been produced and used in many vaccines. Tetanus toxoid is also used both alone and in combination with diphtheria and / or pertussis vaccines. The protein, tetanus toxin, is generally obtained from 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 usually detoxified with formaldehyde and can be purified from the culture filtrate using known methods, such as ammonium sulfate precipitation (see, e.g., Levin and Stone, J. Immunol., 67:235-242 (1951); WHO Manual for the Production and Control of Vaccines: Tetanus Toxoid, 1977 (BLG / UNDP / 77.2 Rev. I.)) or chromatographic techniques, e.g., as disclosed in WO 1996 / 025425. Tetanus toxin may also be inactivated by recombinant genetic means.

[0153] In another embodiment, the carrier protein of the glycoconjugate may be PD (Protein D of Haemophilus influenzae; see, for example, EP0594610B).

[0154] In certain embodiments, a single carrier protein is used in a multivalent pneumococcal conjugate composition. In certain embodiments, more than one protein carrier is used ("hybrid carriers"). These hybrid carrier embodiments can use two, three, four, five, six, seven, eight, nine, or more carrier proteins. Typically, hybrid carrier embodiments include two carrier proteins. For example, in certain embodiments, certain capsular polysaccharides are conjugated to a first protein carrier, and the remaining capsular polysaccharides are attached to a second protein carrier.

[0155] In one embodiment, the first protein carrier is a CRM 197 and the second protein carrier is tetanus toxoid. In certain embodiments, two of the capsular polysaccharides are conjugated to tetanus toxoid and the remaining capsular polysaccharide is CRM 197 In certain embodiments, the two capsular polysaccharides conjugated to tetanus toxoid are selected from the group consisting of serotypes 1, 3, and 5. In certain embodiments, four of the capsular polysaccharides are conjugated to tetanus toxoid, with the remaining capsular polysaccharide being CRM 197 In certain embodiments, the four capsular polysaccharides conjugated to the tetanus toxoid are selected from the group consisting of serotypes 1, 3, 5, 15B, and 22F. In certain embodiments, the four capsular polysaccharides conjugated to the tetanus toxoid are serotypes 1, 5, 15B, and 22F; serotypes 1, 3, 15B, and 22F; or serotypes 3, 5, 15B, and 22F.

[0156] In some embodiments of PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27, capsular polysaccharides from serotypes 1 and 5 are conjugated to tetanus toxoid, and capsular polysaccharides from the remaining serotypes are conjugated to CRM. 197 In another embodiment of PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27, capsular polysaccharides from serotypes 1 and 3 are conjugated to tetanus toxoid, and the remaining capsular polysaccharides are conjugated to CRM 197 In another embodiment of PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27, capsular polysaccharides from serotypes 3 and 5 are conjugated to tetanus toxoid, and the remaining capsular polysaccharides are conjugated to CRM 197 In another embodiment of PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27, capsular polysaccharides from serotypes 1, 5, 15B, and 22F are conjugated to tetanus toxoid, and the remaining capsular polysaccharides are conjugated to CRM 197 PCV-22, PCV-23, PC In another embodiment of PCV-24, PCV-25, PCV-26, or PCV-27, capsular polysaccharides from serotypes 1, 3, 15B, and 22F are conjugated to tetanus toxoid, and the remaining capsular polysaccharides are conjugated to CRM 197 In another embodiment of PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27, capsular polysaccharides from serotypes 3, 5, 15B, and 22F are conjugated to tetanus toxoid, and the remaining capsular polysaccharides are conjugated to CRM 197 It is conjugated to

[0157] Pneumococcal capsular polysaccharides used 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, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B, are described in, for example, WO2006 / 110381, WO2008 / 118752, WO2006 / 110352, and U.S. Patent Application Publication No. 2008 / 0 ... They can be prepared from Streptococcus pneumoniae using any available technique, including standard techniques known to those skilled in the art, such as those disclosed in Patent Publication Nos. 2006 / 0228380, 2006 / 0228381, 2007 / 0184071, 2007 / 0184072, 2007 / 0231340, 2008 / 0102498, and 2008 / 0286838, all of which are incorporated by reference in their entireties. For example, each pneumococcal capsular polysaccharide serotype can be grown in a culture medium (e.g., a soy-based medium). Cells can be lysed, and individual polysaccharides can be purified from the lysate by centrifugation, precipitation, ultrafiltration, and / or column chromatography. Additionally, synthetic protocols can be used to produce pneumococcal capsular oligosaccharides.

[0158] The capsular polysaccharide of Streptococcus pneumoniae comprises repeating oligosaccharide units that can contain up to eight saccharide residues. Capsular saccharide antigens can be full-length polysaccharides or can be reduced in size (e.g., a single oligosaccharide unit or shorter than the original length of the repeating oligosaccharide units). The size of the capsular polysaccharide can be reduced by various methods known in the art, such as acid hydrolysis, hydrogen peroxide treatment, sizing with a high-pressure homogenizer, optionally followed by hydrogen peroxide treatment to generate oligosaccharide fragments, or microfluidization. In certain embodiments, the purified capsular polysaccharide is subjected to a sizing step, such as acid hydrolysis or microfluidization, to reduce its size before reacting it with an oxidizing agent to produce the activated capsular polysaccharide. In certain embodiments, the capsular polysaccharide is not subjected to a sizing step, such as acid hydrolysis or microfluidization, before reacting the purified capsular polysaccharide with an oxidizing agent to produce the activated capsular polysaccharide.

[0159] Pneumococcal conjugates for each serotype can be produced by conjugating the capsular polysaccharide of each serotype to a carrier protein. The different pneumococcal conjugates can be formulated into compositions, including single dose formulations.

[0160] Activation of capsular polysaccharides To produce polysaccharide-protein conjugates, capsular polysaccharides prepared from each pneumococcal serotype can be chemically activated to enable the capsular polysaccharide to react with a carrier protein. Once activated, each capsular polysaccharide can be individually conjugated to a carrier protein to form a glycoconjugate. Chemical activation of the polysaccharide and subsequent conjugation to the carrier protein can be achieved by conventional methods.

[0161] For example, the vicinal hydroxyl groups at the termini of the capsular polysaccharide can be modified by, for example, the methods described in U.S. Pat. Nos. 4,365,170, 4,673,574, and 4,902,506. As disclosed in "Periodates," which are incorporated by reference in their entirety, carbohydrates can be oxidized to aldehyde groups by oxidizing agents such as periodates (including sodium periodate, potassium periodate, or periodic acid). Periodates randomly oxidize vicinal hydroxyl groups of carbohydrates to reactive aldehyde groups, causing C—C bond cleavage. The term "periodate" encompasses both periodate and periodic acid. The term also encompasses metaperiodate (IO 4- ) and orthoperiodate ion (IO6 5- ) The term "periodate" also encompasses various salts of the periodate ion, including sodium periodate and potassium periodate. In certain embodiments, polysaccharides can be oxidized in the presence of sodium metaperiodate.

[0162] In certain embodiments, periodate can be used in an amount of about 0.03 to 0.17 μg per μg of polysaccharide. In certain embodiments, periodate can be used in an amount of about 0.025 to 0.18 μg or about 0.02 to 0.19 μg per μg of polysaccharide. The sugar can be activated as desired within the above range. Outside the range, the effect may be insufficient.

[0163] Polysaccharides may 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.

[0164] For example, cystamine or cysteamine as the spacer can provide thiolated polysaccharides that can be linked to carriers by thioether bonds obtained after reaction with maleimide-activated carrier proteins (e.g., using N-[γ-maleimidobutyryloxy]succinimide ester (GMBS)) or with haloacetylated carrier proteins (e.g., using iodoacetimide, N-succinimidyl bromoacetate (SBA, SIB), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA), or succinimidyl 3-[bromoacetamido]proprionate (SBAP)). Preferably, the cyanate ester (optionally generated by CDAP chemistry) is combined with hexanediamine or adipic acid dihydrazide (AOH), and the amino-derivatized sugar is conjugated to the carrier protein via carboxyl groups on the protein carrier using carbodiimide (e.g., EDAC or EDC) chemistry. Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348, and WO 96 / 129094, all of which are incorporated by reference in their entireties.

[0165] After the activation step, the activated capsular polysaccharide is optionally lyophilized before mixing the activated polysaccharide with the carrier protein. The activated polysaccharide and carrier protein may be lyophilized separately or may be combined with each other and then lyophilized.

[0166] The activated capsular polysaccharide can be lyophilized in the presence of any cryoprotectant, such as a sugar. For example, the sugar may be selected from, but is not limited to, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, and palatinit. In certain embodiments, the sugar is sucrose. The lyophilized polysaccharide is then resuspended in a solvent prior to the conjugation reaction. The lyophilized activated capsular polysaccharide is mixed with a solution containing a carrier protein. Alternatively, the co-lyophilized polysaccharide and carrier protein are resuspended in a solvent prior to the conjugation reaction.

[0167] Conjugation of activated capsular polysaccharide to carrier proteins Conjugation of the activated capsular polysaccharide to the 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, WO2006 / 110381, WO2008 / 079653, and WO2008 / 143709, all of which are incorporated by reference in their entireties. For example, the activated capsular polysaccharide and the carrier protein can be conjugated by reacting them with a reducing agent. Suitable reducing agents include borohydride salts, such as sodium cyanoborohydride, borane-pyridine, sodium triacetoxyborohydride, sodium borohydride, and borohydride exchange resins. At the end of the reduction reaction, unreacted aldehyde groups may remain on the conjugate. The unreacted aldehyde groups can be capped using an appropriate 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 one 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, and 5-ethyl-2-methylpyridine-borane (PEMB).

[0168] The activated capsular polysaccharide may be conjugated directly to the carrier protein or indirectly using 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 carboxylic acid group, two reactive amino groups, or two reactive carboxylic acid groups.

[0169] Other suitable conjugation techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU, as described, for example, in International Patent Application Publication No. WO 98 / 42721, the entire contents of which are incorporated by reference. Conjugation can involve a carbonyl linker, which can be generated by reacting the free hydroxyl group of a sugar with 1,1'-carbonyldiimidazole (CDI) (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 reducing the aromatic terminus to a primary hydroxyl group, optionally protecting / deprotecting the primary hydroxyl group, reacting the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling the CDI carbamate intermediate to an amino group on the protein.

[0170] The polysaccharide to carrier protein ratio for pneumococcal conjugate vaccines is typically in the range of 0.3-3.0 (w / w), but may vary depending on the serotype. The ratio can be determined either by measuring the protein and polysaccharide abundance separately, or by methods known in the art that allow for direct measurement of the ratio. 1 The sugar / protein ratio across the size distribution of the conjugates can be profiled by methods including 1 H NMR spectroscopy or SEC-HPLC-UV / RI with dual monitoring (e.g., refractive index and UV for total material and protein content, respectively), as well as by SEC-HPLC-MALLS or MALDI-TOF-MS. be.

[0171] The resulting polysaccharide-protein conjugates can be purified and enriched by a variety of methods, including concentration / diafiltration, column chromatography, and depth filtration. The purified polysaccharide-protein conjugates are combined to form a multivalent pneumococcal conjugate composition that can be used as a vaccine.

[0172] formulation Formulation of vaccine compositions can be achieved using methods accepted in the industry. Vaccine compositions are formulated to be compatible with their intended route of administration. Individual pneumococcal capsular polysaccharide-protein conjugates 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.

[0173] In some embodiments, the multivalent pneumococcal conjugate composition further comprises an adjuvant. As used herein, "adjuvant" refers to a substance or vehicle that non-specifically enhances the immune response to an antigen. Adjuvants can include, but are not limited to: (1) Aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, aluminum hydroxyphosphate sulfate, etc.; (2) Oil-in-water emulsion formulations (with or without other specific immunostimulants such as muramyl peptides (defined below) or bacterial cell wall components), such as (a) MF59 (WO 90 / 14837) containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally, but not necessarily, containing various amounts of MTP-PE (see below)) formulated into submicron particles using a microfluidizer, such as a Model 110Y Microfluidizer (Microfluidics, Newton, Massachusetts); (b) 10% squalene, 0.4% Tween 80, 5% pluronic-blocked polymer (pluronic-blocked polymer) microfluidized into a submicron emulsion or vortexed to produce a larger particle size emulsion. SAF, containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of 3-O-deacetylated monophosphoryl lipid A (MPL™) (Corixa), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™), as described in U.S. Pat. No. 4,912,094 (Corixa); (3) saponin adjuvants, such as QuilA and STIMULON™ QS-21 (Antigenics, Framingham, Massachusetts) (U.S. Patent No. 5,057,540), or particles produced therefrom, such as ISCOMs (immunostimulating complexes); (4) bacterial lipopolysaccharide, synthetic lipid A analogs, such as aminoalkyl glucosamine phosphate compounds (AGPs) or derivatives or analogs thereof, available from Corixa and described in U.S. Pat. No. 6,113,918; one such AGP is 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl 2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoy]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-bD-glucopyranoside, also known as 529 (formerly known as RC529), formulated in aqueous solution or as a stable emulsion; (5) synthetic polynucleotides, e.g., oligonucleotides containing CpG motifs (U.S. Patent No. 6,207,646); (6) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, etc.; (7) Detoxified mutants of bacterial ADP-ribosylating toxins, such as wild-type or mutant forms of cholera toxin (CT), pertussis toxin (PT), or Escherichia coli heat-labile toxin (LT), in which, for example, glutamic acid at amino acid position 29 has been replaced by another amino acid, preferably histidine, according to WO00 / 18434 (see also WO02 / 098368 and WO02 / 098369), in particular LT-K63, LT-R72, CT-S109, PT-K9 / G129 (see, for example, WO93 / 13302 and WO92 / 19265); and (8) complement components, e.g., trimers of complement component C3d; (9) Biomolecules, such as lipids and costimulatory molecules. Exemplary biological adjuvants include AS04, IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, and 41BBL.

[0174] Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2-(1'-2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamine (MTP-PE), and the like.

[0175] The adjuvant is appropriately selected according to the amount and valence of the conjugate in the composition. In some embodiments, the adjuvant is an aluminum-based adjuvant. When an aluminum-based adjuvant is used, the aluminum in the aluminum-based composition can be added to constitute 0.01 mg / mL to 1 mg / mL. Typically, a single 0.5 ml vaccine dose is formulated to contain approximately 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 between 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 mg 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.

[0176] In certain embodiments, the adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide.

[0177] In a particular embodiment, the adjuvant is aluminum phosphate.

[0178] In some embodiments, the composition is for use as a vaccine against infection with Streptococcus pneumoniae.

[0179] Characterization of pneumococcal capsular polysaccharide-protein carrier conjugates In certain embodiments, the polysaccharide-protein carrier conjugate may have a molecular weight of 100 to 10,000 kDa. In certain embodiments, the conjugate has a molecular weight of 200 to 9,000 kDa. In certain embodiments, the conjugate has a molecular weight of 300 to 8,000 kDa. In certain embodiments, the conjugate has a molecular weight of 400 to 7,000 kDa. In certain embodiments, the conjugate has a molecular weight of 500 to 6,000 kDa. In certain embodiments, the conjugate has a molecular weight of 600 to 5,000 kDa. In certain embodiments, the conjugate has a molecular weight of 500 to 4,000 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0180] When the molecular weight is within the above range, the conjugate can be stably produced with high yield, and the proportion of free polysaccharide can be reduced. In addition, within the above molecular weight range, excellent immunogenicity can be obtained.

[0181] After the individual polysaccharide-protein conjugates are purified, they are combined to formulate the immunogenic compositions of the present disclosure.

[0182] The glyco-protein conjugates of the serotypes of the present disclosure can be characterized by the ratio of polysaccharide to protein carrier (amount of polysaccharide / amount of protein carrier, w / w).

[0183] In certain embodiments, the ratio of polysaccharide to protein carrier (w / w) in the polysaccharide-protein carrier conjugate for each serotype is 0.5 to 2.5, 0.4 to 2.3, 0.3 to 2.1, 0.24 to 2, 0.2 to 1.8, 0.18 to 1.6, 0.16 to 1.4, 0.14 to 1.2, 0.12 to 1, 0.1 to 1, 0.4 to 1.3, 0.5 to 1, or 0.7 to 0.9 (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).

[0184] When the ratio of polysaccharide to protein carrier is within the above range, the conjugate can be stably produced with high yield, and the proportion of free polysaccharide can be reduced. In addition, within the above range, excellent immunogenicity can be obtained, and the conjugate can be kept stable without interference from other serotypes.

[0185] 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 or trapped by the polysaccharide-protein carrier conjugate).

[0186] In certain embodiments, the polysaccharide-protein carrier conjugate contains less than about 60%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15% of the 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% of the 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% of the 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 ... The polysaccharide-protein carrier conjugates of each serotype contain less than about 30% of the free polysaccharides of each serotype, based on the total amount of polysaccharides of each serotype. In certain embodiments, the polysaccharide-protein carrier conjugates of each serotype contain less than about 25% of the free polysaccharides of each serotype, based on the total amount of polysaccharides of each serotype. In certain embodiments, the polysaccharide-protein carrier conjugates of each serotype contain less than about 20% of the free polysaccharides of each serotype, based on the total amount of polysaccharides of each serotype. In certain embodiments, the polysaccharide-protein carrier conjugates of each serotype contain less than about 15% of the free polysaccharides of each serotype, based on the total amount of polysaccharides of each serotype. In certain embodiments, the polysaccharide-protein carrier conjugates of each serotype contain less than about 10% of the free polysaccharides of each serotype, based on the total amount of polysaccharides of each serotype.

[0187] The polysaccharide-protein carrier conjugates of each serotype have a molecular size distribution (K d) can also be characterized. Size exclusion chromatography media (CL-4B; cross-linked agarose beads, 4%) can be used to reveal the relative molecular size distribution of the conjugates. Size exclusion chromatography (SEC) is used in a gravity-fed column to profile the molecular size distribution of the conjugates. Larger molecules, which are excluded from the pores in the media, elute more quickly than smaller molecules. A fraction collector is used to collect the column eluate. Fractions are subjected to colorimetric testing using a sugar assay. K d To determine the fraction at which a molecule is completely excluded, the column is calibrated (V0; K d = 0) and the fraction corresponding to maximum retention (V i ;K d = 1) is defined. The fraction (V e ) is the formula K d =(V e -V0) / (V i -V0) by K d is associated with.

[0188] 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 It can be shown that:

[0189] 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 exhibit 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 exhibit 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 exhibit 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 exhibit a K of 0.3 or less on a CL-4B column. d In certain embodiments, at least 15-60% of the glyco-protein conjugates of each serotype may exhibit a Kd of 0.3 or less on a CL-4B column.

[0190] Prophylactic Methods and Uses In one aspect, the present disclosure provides a vaccine comprising a multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) 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).

[0191] In some embodiments, the vaccine induces a protective immune response in a human subject against disease caused by Streptococcus pneumoniae infection.

[0192] According to a further aspect, the present disclosure provides a method of preventing pneumococcal infection or disease, the method comprising administering to a human subject a prophylactically effective amount of a multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) or a vaccine comprising same. The multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) or a vaccine comprising same can be administered by any route, including, for example, by systemic or mucosal routes, as described in more detail below.

[0193] In certain embodiments, the human subject is an elderly subject, and the disease is pneumonia or invasive pneumococcal disease (IPD). In certain embodiments, the elderly subject is at least 50 years old. In other embodiments, the elderly subject is at least 55 years old. In yet other embodiments, the elderly subject is at least 60 years old.

[0194] In other embodiments, the human subject is a child and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM). In certain embodiments, the child is 0-2 years old. In other embodiments, the child is 2-15 months old.

[0195] 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.

[0196] The amount of conjugate in each vaccine dose or prophylactically effective amount of the mixed carrier multivalent pneumococcal conjugate composition can be selected as an amount that provides protection without significant adverse effects. Such amounts can vary depending on the pneumococcal serotype. Generally, each dose can contain about 0.1 μg to about 100 μg of polysaccharide, particularly about 0.1 to 10 μg, and more particularly about 1 μg to about 5 μg. Optimal amounts of components for a particular vaccine can be determined through standard studies, including observation of appropriate immune responses in subjects. For example, amounts for vaccination of human subjects can be extrapolated from animal studies. Additionally, dosages can be empirically determined.

[0197] In some embodiments, a vaccine or multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) contains about 1 μg to about 5 μg of each capsular polysaccharide; a carrier protein (e.g., CRMP); 197and optionally, about 0.1 mg to about 0.5 mg of elemental aluminum adjuvant. In some embodiments, a vaccine or multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) contains about 2 μg to about 2.5 μg of each capsular polysaccharide, except for serotype 6B and optionally serotype 3, which are present in an amount of about 4 μg to about 5 μg; a protein carrier (e.g., CRMP) 197 ) about 40 μg to about 75 μg; and optionally, about 0.1 mg to about 0.25 mg of elemental aluminum adjuvant.

[0198] In some embodiments, a vaccine or mixed carrier multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) comprises about 1 μg to about 5 μg of each capsular polysaccharide; about 1 μg to about 30 μg of a first carrier protein (e.g., TT); about 1 μg to about 30 μg of a second carrier protein (e.g., CRM); 197 )about and optionally, about 0.1 mg to about 0.5 mg of elemental aluminum adjuvant.

[0199] In some embodiments, a vaccine or mixed carrier multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) comprises about 2 μg to about 2.5 μg of each capsular polysaccharide, except for serotype 6B and optionally serotype 3, which are present in an amount of about 4 μg to about 5 μg; about 2 μg to about 25 μg of a first carrier protein (e.g., TT); about 2 μg to about 25 μg of a second carrier protein (e.g., CRM); 197 ) about 40 μg to about 100 μg; and optionally about 0.1 mg to about 0.25 mg of elemental aluminum adjuvant.

[0200] In some embodiments, the vaccine or multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) 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.

[0201] In some embodiments, a vaccine or multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) may be a single 0.5 ml dose formulated to contain about 2 μg to about 2.5 μg of each capsular polysaccharide, except for 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 multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) may be a single 0.5 ml dose formulated to contain about 2.2 μg of each capsular polysaccharide, except for 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.

[0202] In some embodiments, the vaccine or multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) 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, 15A, 15B, 15C, 18C, 22F, 23A, 23B, 23F, 24F, 33F, and / or 35B, and about 4 μg to about 5 μg of capsular polysaccharides of serotypes 3, 4, 6B, 9V, 19A, and / or 19F.

[0203] In certain embodiments, a vaccine or multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) may be a single 0.5 ml dose formulated to contain about 2 to about 2.5 μg of capsular polysaccharide of serotypes 1, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and / or 35B, and about 4 to about 5 μg of capsular polysaccharide of serotypes 3 and / or 6B.

[0204] In some embodiments, the vaccine or multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) may be a single 0.5 ml dose formulated to contain about 2 to about 2.5 μg of capsular polysaccharide of serotypes 1, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and / or 35B, and about 4 to about 5 μg of capsular polysaccharide of serotype 6B, and / or about 8 to about 9 μg of capsular polysaccharide of serotype 3, more preferably about 8.8 μg of capsular polysaccharide of serotype 3.

[0205] In certain embodiments, the multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) or a vaccine comprising same further comprises sodium chloride and sodium succinate buffer as excipients.

[0206] In some embodiments, the multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) comprises a pneumococcal capsular polysaccharide from serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and / or 35B, each of which is conjugated to a carrier protein (e.g., CRMP). 197 Each 0.5 mL dose contains approximately 2.2 μg of each capsular polysaccharide, except for serotype 6B, which is approximately 4.4 μg; a carrier protein (e.g., CRMP); 197 ) about 40 μg to about 100 μg; about 0.125 to 0.250 mg of elemental aluminum (about 0.5 to about 1.2 mg of aluminum phosphate) as an adjuvant; and sodium chloride and sodium succinate buffers as excipients.

[0207] In some embodiments, a mixed-carrier multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) comprises two or more carrier proteins (mixed carriers). For example, in certain embodiments, at least two serotypes are conjugated to a first carrier protein (e.g., tetanus toxoid) and the remaining serotypes are conjugated to a second carrier protein (e.g., CRM 197In certain embodiments, the two capsular polysaccharides conjugated to the tetanus toxoid are selected from the group consisting of serotypes 1, 3, and 5. In certain embodiments, the two capsular polysaccharides conjugated to the tetanus toxoid are selected from the group consisting of serotypes 1, 3, 5, 15B, and 22F. Instead of, or in addition to, a serotype selected from serotypes 1, 3, 5, 15B, and 22F, one or more of serotypes 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 23A, 23B, 23F, 24F, 33F, and / or 35B may also be conjugated to the tetanus toxoid. Other relevant serotypes may also be conjugated to tetanus toxoid.

[0208] In some embodiments, the mixed carrier multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) comprises pneumococcal capsular polysaccharides from serotypes 1 and 3 each conjugated to TT, and capsular polysaccharides from serotypes 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and / or 35B conjugated to CRM. 197 Each 0.5 mL dose contains about 2.2 μg of each capsular polysaccharide, except for serotype 6B, which contains about 4.4 μg; and about 2 μg to about 25 μg of TT carrier protein. (for serotypes 1 and 3 only), and CRM 197 It can be formulated into a liquid containing about 40 μg to about 100 μg of carrier protein; about 0.125 to 0.250 mg of elemental aluminum (about 0.5 to about 1.2 mg of aluminum phosphate) as an adjuvant; and sodium chloride and sodium succinate buffer as excipients.

[0209] In some embodiments, the mixed carrier multivalent pneumococcal conjugate composition comprises pneumococcal capsular polysaccharides from serotypes 1 and 5 each conjugated to TT, and capsular polysaccharides from serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and / or 35B conjugated to CRM. 197 In one embodiment, each 0.5 mL dose contains about 2.2 μg of each capsular polysaccharide, except for serotype 6B, which is about 4.4 μg, and serotype 3, which is about 2.2 to 8.8 μg; about 2 μg to about 25 μg of TT carrier protein (for serotypes 1 and 5 only); and CRM 197 The vaccine can be formulated into a liquid containing about 40 μg to about 100 μg of carrier protein; about 0.125 to 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 certain embodiments, serotype 3 is present at about 2.2 μg. In other embodiments, serotype 3 is present at about 4.4 μg. In other embodiments, serotype 3 is present at about 8.8 μg. In yet another embodiment, each 0.5 mL dose contains about 2.2 μg of each capsular polysaccharide, except for up to six capsular polysaccharides selected from the group consisting of serotypes 1, 3, 4, 5, 6B, 9V, 19A, and 19F, of which about 4.4 μg is present; about 2 μg to about 25 μg of TT carrier protein (for serotypes 1 and 5 only); and CRM 197The vaccine can be formulated into a liquid containing about 40 μg to about 100 μg of carrier protein; about 0.125 mg to 0.250 mg of elemental aluminum (0.5 mg 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 another embodiment, each 0.5 mL dose contains about 2.2 μg of each capsular polysaccharide except for about 4.4 μg of serotypes 3, 4, 6B, 9V, 19A, and 19F; about 2 μg to about 25 μg of TT carrier protein (for serotypes 1 and 5 only); and CRM 197 The adjuvant may be formulated into a liquid containing about 40 μg to about 100 μg of carrier protein; about 0.125 mg to 0.250 mg of elemental aluminum (0.5 mg 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 2.2 μg of each capsular polysaccharide except for serotypes 3 and 4, which contain about 4.4 μg; about 2 μg to about 25 μg of TT carrier protein (only for serotypes 1 and 5); and CRM 197 It can be formulated into a liquid containing about 40 μg to about 100 μg of carrier protein; about 0.125 mg to 0.250 mg of elemental aluminum (0.5 mg to 1.2 mg aluminum phosphate) adjuvant; and sodium chloride and sodium succinate buffer as excipients.

[0210] In some embodiments, the mixed carrier multivalent pneumococcal conjugate composition comprises pneumococcal capsular polysaccharides from serotypes 3 and 5 each conjugated to TT, and capsular polysaccharides from serotypes 1, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B conjugated to CRM. 197Each 0.5 mL dose contains about 2.2 μg of each capsular polysaccharide except for 6B, which is about 4.4 μg; about 2 μg to about 25 μg of TT carrier protein (for serotypes 3 and 5 only); and CRM 197 Contains about 40 μg to about 100 μg of carrier protein; about 0.125 to 0.250 mg of elemental aluminum (about 0.5 to 1.2 mg of aluminum phosphate) adjuvant; and sodium chloride and sodium succinate buffers as excipients. It can be formulated into a liquid.

[0211] In some embodiments, the mixed carrier multivalent pneumococcal conjugate composition comprises at least two of the pneumococcal capsular polysaccharides of serotypes 1, 3, and 5, and serotypes 15B and 22F, conjugated to tetanus toxoid, with the capsular polysaccharides from the remaining serotypes being conjugated to CRM 197 The compound can be formulated into a liquid formulation in which the compound is conjugated to

[0212] In some embodiments, the mixed carrier multivalent pneumococcal conjugate composition comprises pneumococcal capsular polysaccharides from serotypes 1, 5, 15B, and 22F each conjugated to tetanus toxoid, and capsular polysaccharides from serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 23A, 23B, 23F, 24F, 33F, and 35B conjugated to CRM. 197 Each 0.5 mL dose contains about 2.2 μg of each capsular polysaccharide except for 6B, which is about 4.4 μg; about 2 μg to about 25 μg of TT carrier protein (for serotypes 3 and 5 only); and CRM 197 It can be formulated into a liquid containing about 40 μg to about 100 μg of carrier protein; about 0.125 to 0.250 mg of elemental aluminum (about 0.5 to 1.2 mg of aluminum phosphate) adjuvant; and sodium chloride and sodium succinate buffers as excipients.

[0213] In some embodiments, the mixed carrier multivalent pneumococcal conjugate composition comprises pneumococcal capsular polysaccharides from serotypes 1, 3, 15B, and 22F each conjugated to tetanus toxoid, and capsular polysaccharides from serotypes 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 23A, 23B, 23F, 24F, 33F, and 35B conjugated to CRM. 197 Each 0.5 mL dose contains about 2.2 μg of each capsular polysaccharide except for 6B, which is about 4.4 μg; about 2 μg to about 25 μg of TT carrier protein (for serotypes 3 and 5 only); and CRM 197 It can be formulated into a liquid containing about 40 μg to about 100 μg of carrier protein; about 0.125 to 0.250 mg of elemental aluminum (about 0.5 to 1.2 mg of aluminum phosphate) adjuvant; and sodium chloride and sodium succinate buffers as excipients.

[0214] In some embodiments, the mixed carrier multivalent pneumococcal conjugate composition comprises pneumococcal capsular polysaccharides from serotypes 3, 5, 15B, and 22F each conjugated to tetanus toxoid, and capsular polysaccharides from serotypes 1, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 23A, 23B, 23F, 24F, 33F, and 35B conjugated to CRM. 197 Each 0.5 mL dose contains about 2.2 μg of each capsular polysaccharide except for 6B, which is about 4.4 μg; about 2 μg to about 25 μg of TT carrier protein (for serotypes 3 and 5 only); and CRM 197 It can be formulated into a liquid containing about 40 μg to about 100 μg of carrier protein; about 0.125 to 0.250 mg of elemental aluminum (about 0.5 to 1.2 mg of aluminum phosphate) adjuvant; and sodium chloride and sodium succinate buffers as excipients.

[0215] In some embodiments, the mixed carrier multivalent pneumococcal conjugate composition may be formulated into a liquid formulation in which each of the pneumococcal capsular polysaccharides of serotypes 1 and 5 is conjugated to TT.

[0216] In some embodiments, the mixed carrier multivalent pneumococcal conjugate composition may be formulated into a liquid formulation in which each of the pneumococcal capsular polysaccharides of serotypes 3 and 5 is conjugated to TT.

[0217] In some embodiments, the mixed carrier multivalent pneumococcal conjugate composition may be formulated into a liquid formulation in which each of the pneumococcal capsular polysaccharides of serotypes 1 and 3 is conjugated to TT.

[0218] In some embodiments, the mixed carrier multivalent pneumococcal conjugate composition may be formulated into a liquid formulation in which each of the pneumococcal capsular polysaccharides of serotypes 1, 5, 15B, and 22F is conjugated to TT.

[0219] In some embodiments, the mixed carrier multivalent pneumococcal conjugate composition may be formulated into a liquid formulation in which each of the pneumococcal capsular polysaccharides of serotypes 3, 5, 15B, and 22F is conjugated to TT.

[0220] In some embodiments, the mixed carrier multivalent pneumococcal conjugate composition may be formulated into a liquid formulation in which each of the pneumococcal capsular polysaccharides of serotypes 1, 3, 15B, and 22F is conjugated to TT.

[0221] In some embodiments, the liquid formulation can be filled into a single-dose syringe without preservatives, and after shaking, the liquid formulation becomes a vaccine that is a white homogenous suspension ready for intramuscular administration.

[0222] The multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) may be administered in a single injection or as part of a series of immunizations. For example, the multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) may be administered two, three, four, or more times, spaced at appropriate intervals, e.g., 1, 2, 3, 4, 5, or 6 months apart, or any combination thereof. In some embodiments, a child is administered four doses of a multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) within the first 15 months of life, including, for example, at about 2, 3, 4, and 12-15 months of age; at about 3, 4, 5, and 12-15 months of age; or at about 2, 4, 6, and 12-15 months of age. The first dose may be administered as early as 6 weeks of age. In another embodiment, a child is administered three doses of a multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) within the first 15 months of life, including, for example, at about 2, 4, and 11-12 months of age.

[0223] A multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) 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, as well as those described in International Patent Application WO 02 / 053761.

[0224] A multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) can be administered to a subject by one or more routes of administration known to those of skill in the art, such as parenteral, transdermal or transmucosal, intranasal, intramuscular, intraperitoneal, intradermal, intravenous, or subcutaneous routes, and can be formulated accordingly. A multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) can be formulated to be compatible with its intended route of administration.

[0225] In some embodiments, the multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) can be administered as a liquid formulation by intramuscular, intraperitoneal, subcutaneous, intravenous, intraarterial, or transdermal injection, or by respiratory mucosal injection. The multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) can be formulated in liquid or lyophilized form. In some embodiments, injectable compositions are prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in liquid prior to injection, or emulsions. In some embodiments, injectable solutions and suspensions are prepared from sterile powders or granules. General considerations in the formulation and manufacture of pharmaceuticals administered by these routes can be found, for example, in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Co., Easton, PA, 1995, which is incorporated herein by reference. Currently, oral or nasal sprays or aerosol routes (e.g., by inhalation) are most commonly used for delivering therapeutic agents directly to the lungs and respiratory system. In some embodiments, a multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) 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. No. 4,886,499, U.S. Pat. No. 5,190,521, U.S. Pat. No. 5,328,483, U.S. Pat. No. 5,527,288, U.S. Pat. No. 4,270,537, U.S. Pat. No. 5,015,235, U.S. Pat. No. 5,141,496, U.S. Pat. No. 5,417,662, all of which are incorporated herein by reference.Intradermal compositions may also be administered by a device that limits the effective penetration depth of a needle into the skin, such as that described in WO1999 / 34850 and its functional equivalents, which are incorporated herein by reference.Also suitable is a jet injection device, which delivers liquid vaccine to the dermis by a liquid jet injector or a needle that generates a jet that penetrates the stratum corneum and reaches the dermis.Jet injection devices are, for example, described 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 No. 5,312,335, U.S. Patent No. 5,503,627, U.S. Patent No. 5,064,413, U.S. Patent No. 5,520,639, U.S. Patent No. 4,596,556, U.S. Patent No. 4,790,824, U.S. Patent No. 4,941,880, U.S. Patent No. 4,940,460, WO1997 / 37705, and WO1997 / 13537 (all of which are incorporated herein by reference). Also suitable is a ballistic powder / particle delivery device, which uses compressed gas to accelerate the powdered vaccine through the outer skin layer into the dermis.In addition, a conventional syringe can be used in the classical Mantoux method of intradermal administration.

[0226] Products for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include 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 fish oil, and lipids obtained 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 solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, etc. or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as, for example, antibacterial agents, antioxidants, chelating agents, and inert gases and the like.

[0227] The multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) can be formulated in the form of a unit-dose vial, a multi-dose vial, or a pre-filled syringe. Pharmaceutically acceptable carriers for solutions include aqueous or non-aqueous solvents, suspensions, emulsions, or oils. The composition can be isotonic, hypertonic, or hypotonic. However, it is desirable for compositions for infusion or injection to be essentially isotonic. Therefore, isotonicity or hypertonicity may be advantageous for the preservation of the composition. If the composition is hypertonic, it can be diluted to isotonicity before administration. The tonicity agent can be an ionic tonicity agent such as a salt or a non-ionic tonicity 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 glycerin. Preferably, at least one pharmaceutically acceptable buffer is included. For example, when the composition is an infusion or injection solution, it is preferably formulated in a buffer having a buffering capacity of pH 4 to pH 10, such as pH 5 to pH 9 or pH 6 to pH 8. The buffer can be selected from those appropriate for the United States Pharmacopeia (USP). For example, the buffer can be selected from the group consisting of monobasic acids such as acetic acid, benzoic acid, gluconic acid, and 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.

[0228] The multivalent pneumococcal conjugate compositions (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) may contain 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 various repeating ethoxy (oxy-1,2-ethanediyl) groups, particularly octoxynol-9 (Triton-100); ethylphenoxypolyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as lecithin; and TERGITOL. Nonylphenol ethoxylates such as the NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl, and oleyl alcohols (Brij surfactants), especially triethylene glycol monolauryl ether (Brij 30); sorbitan ethers known as SPAN, especially sorbitan trioleate (Span 85) and sorbitan monolaurate.

[0229] A mixture of surfactants can be used, such as Tween 80 / Span 85. A combination of polyoxyethylene sorbitan esters, such as Tween 80 and Triton Octoxynols such as X-100 are also suitable. A combination of Laureth 9 and Tween and / or octoxynols 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 (e.g., Triton X-100) included is 0.001% to 0.1% (w / v), in particular 0.005% to 0.02%. The amount of polyoxyethylene ether (e.g., Laureth 9) present may be 0.1% to 20% (w / v), perhaps 0.1% to 10%, particularly 0.1% to 1% or about 0.5%.

[0230] In some embodiments, the multivalent pneumococcal conjugate composition (e.g., PCV-22, PCV-23, PCV-24, PCV-25, PCV-26, or PCV-27) can be delivered via a controlled release system. For example, intravenous infusion, transdermal patch, liposomes, or other routes can be used for administration. In one aspect, polymers or implants such as microspheres can be used.

[0231] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention. [Example]

[0232] Production of pneumococcal capsular polysaccharides Cultivation of pneumococci and purification of capsular polysaccharides were performed as known to those skilled in the art. Pneumococcal 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 23B: ATCC No. 10364; serotype 23F: ATCC No. 6323). For serotypes 8, 10A, 11A, 12F, 15A, 15B, 15C, 22F, 23A, 23B, 24F, 33F, and 35B, internal strain(s) obtained from other sources were used, although any publicly available strain can be used. Pneumococci were characterized by capsule and motile, Gram-positive, lancet-shaped diplococci, and alpha hemolysis on blood agar. Serotypes were identified by a swelling test using specific antisera (U.S. Patent No. 5,847,112).

[0233] Cell bank production To expand the strain and remove animal-derived components, several generations of seed stocks were generated (generations F1, F2, and F3). Two further generation seed stocks were generated. The first further generation was cultured from the F3 vial, and subsequent generations were cultured from the first further generation vial. The seed vials were stored frozen (below -70°C) with synthetic glycerol as a cryopreservative. For cell bank production, 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).

[0234] Culture and harvest Cultures obtained 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 terminating the cultivation, sodium deoxycholate was added to the culture to lyse the cells. The resulting fermenter contents were cooled to induce protein precipitation. The mixture was then centrifuged to remove precipitated proteins and cell debris.

[0235] purification 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 using 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 precipitate polysaccharides, and the supernatant was collected. The filtrate was concentrated using a 30 kDa membrane, and the concentrate was diafiltered with approximately 10 volumes of triple-distilled water. After diafiltration, the remaining solution was filtered through a 0.2 μm filter. The filtrate was subjected to in-process control tests (appearance, residual protein, residual nucleic acid, endotoxin, molecular weight, and total polysaccharide). The concentrate was sterile filtered and stored at -20°C. [Example]

[0236] Production of conjugates of pneumococcal capsular polysaccharides and carrier proteins (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F) The polysaccharides of the various serotypes are activated according to different pathways and then conjugated to carrier proteins, CRMs, 197 Specifically, as disclosed below, each of the capsular polysaccharides of serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F was conjugated to CRMP. 197Multivalent pneumococcal polysaccharide-protein conjugates containing capsular polysaccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B were produced by conjugating each of the capsular polysaccharides of serotypes 1 and 5 to CRM. 197 Conjugation to serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B is described in Examples 3 to 8. Additional multivalent pneumococcal polysaccharide-protein conjugates comprising capsular polysaccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, and 33F are prepared by conjugating each of the capsular polysaccharides of serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, and 33F to CRMs, as disclosed below. 197 and by conjugating each of the capsular polysaccharides of serotypes 1, 5, 15B and 22F to TT.

[0237] It is also contemplated that serotype 3 may be conjugated to TT instead of, or in addition to, serotypes 1 or 5 as disclosed in WO2019 / 152925. 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 by ultrafiltration.

[0238] The polysaccharides of the various serotypes are activated according to the following different pathways and then attached to the carrier protein, CRM. 197 Specifically, each of the capsular polysaccharides of all serotypes except 15B and 22F was conjugated to CRM 197Conjugates were produced 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 by ultrafiltration. The conjugates were purified using ultrafiltration and finally filtered through a 0.2 μm filter. Process parameters such as pH, temperature, concentration, and time were as follows:

[0239] (1) Activation process Step 1: Hydrolysis Reductive amination is a known method for conjugating polymers in which an amide bond is formed between the primary amine (-NH2) group of the protein and the aldehyde of the sugar. Aldehyde groups are added to pneumococcal capsular polysaccharides to facilitate conjugation to carrier proteins. Vicinal diol units of monosaccharides can be oxidized with sodium periodate (NaIO4) to form aldehyde groups. Capsular polysaccharides from serotypes 1, 3, 4, 6A, 8, 11A, 12F, 14, 15B, 18C, 22F, and 33F were pretreated as follows.

[0240] For serotype 1, sodium hydroxide was added to the capsular polysaccharide solution (for a final base concentration of 0.05 M) and the solution was incubated at 50±2° C. The solution was then cooled to a temperature ranging from about 21° C. to about 25° C., and hydrochloric acid was added to it to a final pH of 6.0±0.1, thereby stopping the hydrolysis.

[0241] For serotypes 3, 8, 11A, and 15B, hydrochloric acid was added to the capsular polysaccharide solution (to a final acid concentration of 0.01 M), and the solution was incubated at 60±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 it to a final pH of 6.0±0.1, thereby stopping the hydrolysis.

[0242] For serotype 4, hydrochloric acid was added to the capsular polysaccharide solution (to a final acid concentration of 0.1 M) and the solution was incubated at 45±2° C. It was then cooled to a temperature ranging from about 21° C. to about 25° C., and hydrochloric acid was added to it to a final pH of 6.0±0.1, thereby stopping the hydrolysis.

[0243] For serotype 6A, glacial acetic acid was added to a solution of capsular polysaccharide (to a final acid concentration of 0.1 M) and the solution was incubated at 60±2° C. The solution was then cooled to a temperature ranging from about 21° C. to about 25° C., and 1 M sodium phosphate was added to it to a final pH of 6.0±0.1, thereby stopping the hydrolysis.

[0244] For serotype 12F, hydrochloric acid was added to a solution of capsular polysaccharide (to a final acid concentration of 0.01 M) 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 thereto to a final pH of the solution of 6.0±0.1, thereby stopping the hydrolysis.

[0245] For serotypes 14 and 18C, glacial acetic acid was added to the capsular polysaccharide solution (to a final acid concentration of 0.2 M), and the solution was incubated at 94±2° C. The solution was then cooled to a temperature ranging from about 21° C. to about 25° C., and 1 M sodium phosphate was added thereto so that the final pH of the solution was 6.0±0.1, thereby stopping the hydrolysis.

[0246] For serotypes 22F and 33F, hydrochloric acid was added to the capsular polysaccharide solution (to a final acid concentration of 0.01 M) and the solution was incubated at 60±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 it to a final pH of 6.0±0.1, thereby stopping the hydrolysis.

[0247] Each of the resulting capsular polysaccharides was diluted with water for injection (WFI), sodium acetate, and sodium phosphate to a final concentration of between about 1.0 mg / mL and about 2.0 mg / mL.

[0248] Step 2: Periodate reaction The sodium periodate molar equivalent for each pneumococcal sugar activation was determined based on the repeating unit molar mass. For all serotypes except 1, 7F, and 19F, thoroughly mixed The oxidation reaction proceeded for 16-20 hours at 21-25°C while the temperature was below 10°C. To help maintain consistent and stable production of conjugate, a range of degree of oxidation (Do) levels for each serotype was targeted during the conjugation process. The preferred targeted range of Do levels for each serotype is shown in Tables 1 and 2.

[0249] [Table 1]

[0250] [Table 2]

[0251] Step 3: Ultrafiltration The oxidized sugars were concentrated and diafiltered with WFI through 100 kDa MWCO ultrafiltration membranes (30 kDa for serotype 1 and 5 kDa 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 filtrate was discarded, and the retentate was filtered through a 0.2 μm filter.

[0252] Step 4: Freeze drying For capsular polysaccharides of serotypes 3, 4, 5, 8, 9N, 9V, 10A, 14, and 33F to be 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.

[0253] For the capsular polysaccharides of serotypes 1 and 18C to be 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.

[0254] For capsular polysaccharides of serotypes 6A, 6B, 7F, 15B-TT, 19A, 19F, 22F-TT, and 23F to be conjugated to carrier proteins by using DMSO solvent, a predetermined amount of sucrose was added to the activated saccharide to reach a final sucrose concentration of 5% ± 3% (w / v), and the samples were prepared independently, lyophilized, and then stored at -25°C ± 5°C.

[0255] For serotype 11A capsular polysaccharide, the activated saccharide was supplemented with a predetermined amount of sucrose 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.

[0256] For serotype 12F capsular polysaccharide, the activated sugar was supplemented with a predetermined amount of sucrose 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.

[0257] (2) Conjugation process Aqueous conjugation of serotypes 1, 3, 4, 5, 8, 9N, 9V, 10A, 14, 18C, and 33F was performed, and DMSO conjugation of serotypes 6A, 6B, 7F, 11A, 12F, 15B-TT, 19A, 19F, 22F-TT, and 23F was performed. Each capsular polysaccharide was conjugated to the carrier protein at a ratio of 0.2 to 2:1.

[0258] Step 1: Melting Aqueous conjugation For serotypes 1, 3, 4, 5, 8, 9N, 9V, 10A, 14, 18C, and 33F, lyophilized samples were thawed and equilibrated at room temperature. The lyophilized samples were reconstituted to reaction concentrations using sodium phosphate buffer solution at 23 ± 2°C for each serotype ratio set.

[0259] Dimethyl sulfoxide (DMSO) conjugation 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 with DMSO.

[0260] Step 2: Conjugation reaction Aqueous conjugation For serotypes 3-TT, 4, 5-TT, 8, 9N, 9V, 10A, 14, 18C, and 33F, the conjugation reaction was initiated by adding 1.0 to 1.4 moles of sodium cyanoborohydride per mole of sugar in a solution (100 mg / mL). However, for serotypes 1, 1-TT, and 3, the conjugation reaction was initiated by adding 0.5 moles of sodium cyanoborohydride per mole of sugar in a solution (100 mg / mL). The reaction was initiated by the addition of

[0261] 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 achieve 1.8 to 2.2 molar equivalents of sodium borohydride per mole of sugar. The mixture was incubated at 23±2°C for 3 to 6 hours. This procedure reduced any unreacted aldehydes present on the sugar. 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.

[0262] DMSO conjugation 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 saccharide. WFI was added to the reaction mixture to a target concentration of 1% (v / v), and the mixture was incubated at 23 ± 2 °C for 12–26 h. A 100 mg / mL sodium borohydride solution (typically 1.8–2.2 molar equivalents of sodium borohydride per mole of activated saccharide) and WFI (target 5% v / v) were added to the reaction, and the mixture was incubated at 23 ± 2 °C for 3–6 h. This procedure reduced any unreacted aldehydes present on the saccharide. The reaction mixture was then diluted with sodium chloride 0.9% and the diluted conjugation mixture was filtered using a 0.8 or 0.45 μm prefilter.

[0263] Step 3: Ultrafiltration The diluted conjugate mixture was concentrated and diafiltered through a 100 kDa MWCO or 300 kDa MWCO ultrafilter with a minimum of 15 volumes of 0.9% sodium chloride or buffer, the composition and pH of which varied depending on the serotype.

[0264] Step 4: Sterile filtration The retentate after ultrafiltration was sterile filtered (0.2 μm) and in-process controls were performed on the filtered conjugate (appearance, free protein, free sugars, molecular size distribution, sterility, sugar content, protein content, pH, endotoxin, residual cyanide, residual DMSO, sugar identity, TT identity and CRM). 197 The final concentrate was stored refrigerated at 2-8°C.

[0265] CRMs of serotypes 15A, 15C, 23A, 23B, 24F, and 35B 197 Conjugation to is described in Examples 3-8. [Example]

[0266] Serotype 15A and CRM 197 Preparation of monoconjugates of Serotype 15A polysaccharide can be purified as discussed above or by referring to the method described in WO2013 / 191459 for purifying polysaccharides of other serotypes. Acid hydrolysis was performed by applying acid and heat to the purified serotype 15A polysaccharide as shown in Table 1, followed by an activation process. It was observed that the hydrolysis conditions affected the degree of oxidation (Do) and molecular weight of the activated polysaccharide, as well as the conjugation results. The activation and conjugation processes were carried out under the same conditions. Sodium periodate was added, and the oxidation reaction was carried out at 21-25°C for 16-20 hours. The activated polysaccharide and CRM 197 The protein was lyophilized and suspended in DMSO. The reaction concentration was 1.5 mg / mL based on polysaccharide content, and the activated polysaccharide and protein were added. The proteins were mixed in a 1:1 ratio. Cyanoborohydride was added to initiate the conjugation reaction, and the mixture was incubated at 23°C ± 2°C for 20-28 hours. The borohydride solution mixture was incubated at 23°C ± 2°C for 3-6 hours. This process reduced any unreacted aldehydes present in the sugar, followed by concentration and dialysis using an ultrafiltration filter.

[0267] [Table 3]

[0268] Serotype 15A and CRM 197 The effect of oxidation level (Do) on the conjugation of 15A polysaccharide was evaluated. 0.1 M HCl was added to the 15A polysaccharide and incubated at 60°C for 90 minutes. The amount of sodium periodate was adjusted, and the oxidation reaction was carried out at 21-25°C for 16-20 hours. The activated polysaccharide and CRM 197The protein was lyophilized and suspended in DMSO. Activated polysaccharide and protein were mixed in a 1:1 ratio, with a reaction concentration of 1.5 mg / mL based on polysaccharide content. Conjugation with cyanoborohydride was carried out as described above when assessing the effect of acid hydrolysis on serotype 15A.

[0269] [Table 4]

[0270] The effect of the polysaccharide to protein reaction ratio on conjugation was also evaluated. 197 The protein was lyophilized and suspended in DMSO. The activated polysaccharide and protein were mixed at the ratios listed in Table 5, with a reaction concentration of 1.0 mg / mL based on polysaccharide content, and conjugation with cyanoborohydride was performed when assessing the effect of acid hydrolysis on serotype 15A.

[0271] [Table 5] [Example]

[0272] Serotype 15C and CRM 197 Preparation of monoconjugates of Serotype 15C polysaccharide can be purified as discussed above or by referring to the method described in WO2013 / 191459 for purifying polysaccharides of other serotypes. The amount of sodium periodate added to the 15C polysaccharide was adjusted, and the oxidation reaction was carried out at 21-25°C for 16-20 hours. Activated polysaccharides and CRM 197The protein was lyophilized and suspended in phosphate buffer. The activated polysaccharide and protein were mixed in a 1:1 ratio, with the reaction concentration based on polysaccharide content being 1.5 mg / mL. Cyanoborohydride was added to initiate the conjugation reaction, and the mixture was incubated at 37°C ± 2°C for 44-52 hours. The borohydride solution mixture was incubated at 23°C ± 2°C for 3-6 hours. This process reduced any unreacted aldehydes present in the sugar, followed by concentration and dialysis using an ultrafiltration filter.

[0273] [Table 6]

[0274] Serotype 15C and CRM using DMSO 197 The effect of oxidation level (Do) on the conjugation of 15C polysaccharide was evaluated. The amount of sodium periodate added to the 15C polysaccharide was adjusted, and the oxidation reaction was carried out at 21-25°C for 16-20 hours. 197 The protein was lyophilized and suspended in DMSO. The activated polysaccharide and protein were mixed in a 1:1 ratio, with the reaction concentration based on polysaccharide content being 1.5 mg / mL. Cyanoborohydride was added to initiate the conjugation reaction, and the mixture was incubated at 23°C ± 2°C for 20-28 hours. The borohydride solution mixture was incubated at 23°C ± 2°C for 3-6 hours. This process reduced any unreacted aldehydes present in the sugar, followed by concentration and dialysis using an ultrafiltration filter.

[0275] [Table 7] [Example]

[0276] Serotype 23A and CRM 197 Preparation of monoconjugates of Serotype 23A polysaccharides can be purified as discussed above or by referring to the methods described in WO2013 / 191459 for purifying polysaccharides of other serotypes. To evaluate the effect of the degree of oxidation (Do) on conjugation, the amount of sodium periodate was adjusted and the oxidation reaction was carried out at 21-25°C for 16-20 hours. Activated polysaccharides and CRMs 197 The protein was lyophilized and suspended in DMSO. The activated polysaccharide and protein were mixed in a 1:1 ratio, with a reaction concentration of 1 mg / mL based on polysaccharide content. Alternatively, the activated polysaccharide and protein were mixed in the ratios listed in Table 8, with a reaction concentration of 1.5 mg / mL based on polysaccharide content. Cyanoborohydride was added to initiate the conjugation reaction, and the mixture was incubated at 23°C ± 2°C for 20-28 hours. The borohydride solution mixture was incubated at 23°C ± 2°C for 3-6 hours. This process reduced any unreacted aldehydes present in the sugar, followed by concentration and dialysis using an ultrafiltration filter. The effect of varying Do levels on conjugation is shown in Table 8.

[0277] [Table 8]

[0278] The effect of polysaccharide to protein reactivity ratio on conjugation is shown in Table 9.

[0279] [Table 9]

[0280] Serotype 23A and CRM 197 The effect of reaction concentration on the conjugation between purified serotype 23A polysaccharide was evaluated using sodium periodate as discussed above. The activated polysaccharide and CRM 197The protein was lyophilized and suspended in DMSO. The activated polysaccharide and protein were mixed in a 1:1 ratio, with reaction concentrations based on polysaccharide content as listed in Table 10, and conjugation with cyanoborohydride was carried out as described above when assessing the effect of Do on serotype 23A.

[0281] [Table 10]

[0282] Serotype 23A and CRM 197 The effect of acid hydrolysis on the conjugation between the purified serotype 23A polysaccharide and CRM was evaluated. Acid hydrolysis was performed by applying acid and heat to the purified serotype 23A polysaccharide as shown in Table 11, followed by an activation process. The activation and conjugation processes were carried out under the same conditions. Sodium periodate was added, and the oxidation reaction was carried out at 21-25°C for 16-20 hours. The activated polysaccharide and CRM 197 The protein was lyophilized and suspended in phosphate buffer. The activated polysaccharide and protein were mixed in a 1:1 ratio, with the reaction concentration based on polysaccharide content being 1.5 mg / mL. Cyanoborohydride was added to initiate the conjugation reaction, and the mixture was incubated at 37°C ± 2°C for 44-52 hours. The borohydride solution mixture was incubated at 23°C ± 2°C for 3-6 hours. This process reduced any unreacted aldehydes present in the sugar, followed by concentration and dialysis using an ultrafiltration filter.

[0283] [Table 11]

[0284] Serotype 23A and CRM 197 The effect of using phosphate buffer on the conjugation between activated serotype 23A polysaccharide and CRM was evaluated. The amount of sodium periodate was adjusted to activate serotype 23A, and the oxidation reaction was carried out at 21–25°C for 16–20 h.197 The protein was lyophilized and suspended in phosphate buffer. The activated polysaccharide and protein were mixed in a 1:1 ratio, with the reaction concentration being 15 mg / mL based on polysaccharide content. Cyanoborohydride was added to initiate the conjugation reaction, and the mixture was incubated at 23°C ± 2°C for 20-28 hours. The borohydride solution mixture was incubated at 23°C ± 2°C for 3-6 hours. This process reduced any unreacted aldehydes present in the sugar, followed by concentration and dialysis using an ultrafiltration filter.

[0285] [Table 12] [Example]

[0286] Serotype 23B and CRM 197 Preparation of monoconjugates of Serotype 23B polysaccharides can be purified as discussed above or by referring to the methods described in WO2013 / 191459 for purifying polysaccharides of other serotypes. To evaluate the effect of the degree of oxidation (Do) on conjugation, the amount of sodium periodate used to activate serotype 23B was adjusted, and the oxidation reaction was carried out at 21-25°C for 16-20 hours. The activated polysaccharides and CRM 197 The protein was lyophilized and suspended in DMSO. The activated polysaccharide and protein were mixed in a 1:1 ratio, with a reaction concentration of 1.5 mg / mL based on polysaccharide content. Alternatively, the amount of sodium periodate was kept constant, with the activated polysaccharide and protein mixed in the ratios listed in Table 13, with a reaction concentration of 1.5 mg / mL based on polysaccharide content. Cyanoborohydride was added to initiate the conjugation reaction, and the mixture was incubated at 23°C ± 2°C for 20-28 hours. The borohydride solution mixture was incubated at 23°C ± 2°C for 3-6 hours. This process reduced any unreacted aldehydes present in the sugars, followed by concentration and dialysis using an ultrafiltration filter. The effect of varying Do levels on conjugation is shown in Table 13.

[0287] [Table 13]

[0288] The effect of polysaccharide to protein reactivity ratio on conjugation is shown in Table 14.

[0289] [Table 14] [Example]

[0290] Serotypes 24F and CRM 197 Preparation of monoconjugates of Serotype 24F polysaccharide can be purified as discussed above or by referring to the methods described in WO2013 / 191459 for purifying polysaccharides of other serotypes. The purified serotype 24F polysaccharide was subjected to acid hydrolysis or microfluidization. Sodium periodate was then added to the serotype 24F polysaccharide, and the oxidation reaction was carried out at 21-25°C for 16-20 hours. The activated polysaccharide and CRM 197 The protein was lyophilized and suspended in phosphate buffer. The activated polysaccharide and protein were mixed in a 1:1 ratio, with the reaction concentration being 10 mg / mL based on polysaccharide content. Cyanoborohydride was added to initiate the conjugation reaction, and the mixture was incubated at 37°C ± 2°C for 44-52 hours. The borohydride solution mixture was incubated at 23°C ± 2°C for 3-6 hours. This process reduced any unreacted aldehydes present in the sugar, followed by concentration and dialysis using an ultrafiltration filter. The molar equivalents of cyanoborohydride and borohydride were as listed in Table 15. Substitutes for capping reagent (borohydride) were used. Cyanoborohydride activated 24F polysaccharide and CRM 197 Addition of excess capping reagent (borohydride) to the protein improves the quality of the conjugate as indicated by an increase in the molecular weight of the conjugate. Addition of excess capping reagent (borohydride) improves the quality of the conjugate as indicated by a decrease in the molecular weight of the conjugate. 197 It had a negative effect on the conjugate.

[0291] [Table 15] [Example]

[0292] Serotype 35B and CRM 197 Preparation of monoconjugates of Serotype 35B can be purified as discussed above or by referring to the methods described in WO 2013 / 191459 for purifying polysaccharides of other serotypes. Serotype 35B polysaccharide was diluted with distilled water (DW) to a final concentration of 1.0 mg / mL to 2.0 mg / mL.

[0293] Periodate reaction. To evaluate the effect of degree of oxidation (Do) on conjugation, the amount of sodium periodate used to activate serotype 35B was adjusted, and the oxidation reaction was carried out at 21–25 °C for 16–20 h. Molar equivalents of 0.007–0.15 sodium periodate relative to the polysaccharide content were used.

[0294] Ultrafiltration. The activated serotype 35B polysaccharide was concentrated and diafiltered using a 30 kDa MWCO ultrafiltration filter with DW. The filtered product was discarded and the retentate was filtered through a 0.22 μm filter.

[0295] Lyophilization. The specified amount of sucrose, calculated to reach a 5% ± 3% sucrose concentration, was added to the activated serotype 35B polysaccharide. Concentrated sugars and CRM 197The carrier proteins were each filled into a vial and lyophilized. Alternatively, the activated serotype 35B polysaccharide and the carrier protein were mixed, filled into a glass bottle, and lyophilized.

[0296] Dissolution. Lyophilized activated serotype 35B saccharide and lyophilized CRM 197 The carrier protein was equilibrated at room temperature. Activated serotype 35B saccharide was resuspended in phosphate buffer at a concentration of 12.5 g / L to 17.5 g / L saccharide. The pH of the phosphate buffer for the conjugation reaction was adjusted to pH 6.0 to pH 7.2. At this point, the carrier protein was used at a concentration of 6.25 g / L to approximately 35 g / L (corresponding to a PR:PS weight ratio of 1:0.5 to 2).

[0297] Conjugation reaction. The conjugation reaction was initiated by adding sodium cyanoborohydride solution (100 mg / mL) at a ratio of 1.0–1.4 molar equivalents per mole of activated sugar. The mixture was incubated at 37 ± 2 °C for 44–52 h. The reaction material was then added to 100 mg / mL of sodium borohydride solution (typically 1.0–1.4 molar equivalents per mole of activated sugar). 8 to 2.2 molar equivalents of sodium borohydride were added, and the mixture was incubated at 23°C ± 2°C for 3 to 6 hours. This process reduced any unreacted aldehydes present in the sugar, followed by concentration using an ultrafiltration filter and dialysis. The reaction mixture was then diluted with 0.9% sodium chloride, and the diluted conjugate was filtered through a 0.45 μm filter.

[0298] Ultrafiltration: The diluted conjugate mixture was concentrated and diafiltered with at least 20 volumes of 0.9% sodium chloride solution or buffer using a 100 kDa MWCO ultrafiltration filter. The filtered product was discarded.

[0299] Sterile filtration. The retentate after diafiltration was filtered through a 0.22 μm filter. Filtered product 35B-CRM 197In-process controls (sugar content, free protein, free sugars, and residual cyanide) were performed on the conjugate. In-process controls were performed on the filtered retentate to determine if further concentration, diafiltration, and / or dilution was required. If necessary, the filtered conjugate was diluted with 0.9% sodium chloride to a final concentration of less than 0.55 g / L. At this stage, tests were performed for sugar content, protein content, and sugar:protein ratio. The conjugate was filtered (0.22 μm filter) and subjected to release studies (appearance, free protein, free sugars, endotoxins, molecular sizing, residual cyanide, sugar identity, and CRM). 197 The serotype 35B glycoconjugates contained at least 0.2 mM acetate per mM 35B polysaccharide. The final conjugate concentrate was refrigerated at 2-8°C. The analytical results of several representative preparations of serotype 35B glycoconjugates are shown in Table 16 below.

[0300] [Table 16]

[0301] As can be seen in Table 16, the methods described herein for making serotype 35B glycoconjugates show good conjugation yields, allowing for the production of conjugates with low % free sugar and good stability. [Example]

[0302] Serotype-specific IgG concentration measurement Serotype 15A-CRM produced in Example 3 197 Monoconjugate, serotype 15C-CRM prepared in Example 4 197 Monoconjugate, serotype 23A-CRM prepared in Example 5 197 Monoconjugate, serotype 23B-CRM prepared in Example 6 197 Monoconjugate, serotype 24F-CRM prepared in Example 7 197Monoconjugate and serotype 35B-CRM prepared in Example 8 197 The monoconjugate was tested for its ability to induce an immunogenic response in rabbits. Immunogenicity assessment was performed by antigen-specific ELISA for serum IgG concentration and by oposomal assay (OPA) for antibody functionality. New Zealand White rabbits were immunized intramuscularly at weeks 0 and 2 with a human dose (2.2 μg of polysaccharide). Serum was sampled every two weeks after immunization.

[0303] Capsular polysaccharides (PnPs) of serotypes 15A, 15C, 23A, 23B, 24F, and 35B were coated onto 96-well plates at 0.5 to 1 μg / well. Equal amounts of serum were sampled from each subject and pooled by group. The plates were washed with wash buffer and incubated with blocking buffer at 37°C for 1 hour. The serum pool was serially diluted 2.5-fold with 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 plates were washed five times with wash buffer, and then 50 μl of the adsorbed, diluted serum was added to the coated wells and incubated at room temperature for 2 to 18 hours. The well plate was washed in the same manner, and then 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 of p-nitrophenylamine buffer was added to each well as a substrate, followed by incubation at room temperature for 2 hours. The reaction was quenched by adding 50 μl of 3 M NaOH, and the absorbance was measured at 405 nm and 690 nm. The results are shown in Table 17.

[0304] [Table 17]

[0305] For serotype 35B, IgG concentrations were measured from different groups of glycoconjugates based on the pH of the conjugation reaction and the molecular weight of the 35B glycoconjugate as shown in Table 18.

[0306] [Table 18]

[0307] Functional Immunogenicity Assay (MOPA) for Monovalent Conjugates Antibody function was assessed by testing serum in the MOPA assay. Pneumococcal MOPA strains stored at -70°C or below were diluted to the corresponding final dilution factor so that each strain had a concentration of approximately 50,000 CFU / mL. Equal amounts of serum were sampled from each subject, pooled by group, and serially diluted two-fold to leave 20 μl of serum in a U-bottom plate. After dilution, 10 μl of the strain prepared for each serotype was mixed with the diluted sample. The mixture was incubated at room temperature for 30 minutes to ensure sufficient mixing of the pneumococci and antibodies. A mixture of pre-differentiated 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 a THY agar plate that had been pre-dried for 30–60 minutes. The plate was then allowed to absorb for 20 minutes until dry. A 25 mg / mL TTC stock solution was added to the prepared overlay agar, and the appropriate antibody for the corresponding strain was added to it. The mixture was mixed thoroughly, and then approximately 25 mL of the mixture was added to the plate and allowed to harden for approximately 30 minutes. The fully hardened plate was incubated in a CO2 incubator (37°C) for 12 to 18 hours, and then the colonies were counted. The MOPA titer was expressed as the dilution at which 50% killing was observed. The results are shown in Table 19.

[0308] [Table 19]

[0309] For serotype 35B, the conjugation reaction pH and 3 as shown in Table 20 Based on the molecular weight of the 5B glycoconjugates, MOPA titers were determined for different groups of glycoconjugates.

[0310] [Table 20] [Example]

[0311] Formulation of a 27-valent pneumococcal conjugate vaccine with polysaccharides from serotypes 1 and 5 conjugated to tetanus toxoid Based on the batch volume and bulk sugar concentration, the desired volume of the final bulk concentrate from Examples 2-8 was calculated. 0.85% sodium chloride (saline), polysorbate 80, and succinate buffer were added to a pre-labeled formulation container, followed by the bulk concentrate. The product was then mixed thoroughly 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 if necessary. The formulated bulk product was stored at 2-8°C. The following multivalent pneumococcal conjugate vaccine formulation was manufactured and designated PCV27-(1 / 5)-TT:

[0312] PCV27(1 / 5)-TT contains polysaccharides of serotypes 1 and 5 in TT and polysaccharides of serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B in CRM. 197 and polysaccharide conjugates prepared by conjugating to a

[0313] PCV27(1 / 5)-TT in a total dose of 0.5 ml contains 2.2 μg of each saccharide except for serotype 6B, which contains 4.4 μg, approximately 2 μg to 25 μg of TT (for serotypes 1 and 5), and approximately 45 μg to 100 μg of CRM. 197, 0.125 mg of elemental aluminum (0.5 mg aluminum phosphate) adjuvant, 4.25 mg of sodium chloride, approximately 295 μg of succinate buffer solution, and approximately 120 μg of polysorbate 80. [Example]

[0314] Immunogenicity of the multivalent pneumococcal conjugate vaccine (PCV27(1 / 5)-TT) The composite carrier, multivalent pneumococcal vaccine, PCV27(1 / 5)-TT, prepared in Example 10, was tested for its ability to induce an immunogenic response in rabbits. Serum IgG concentrations were measured by antigen-specific ELISA, and antibody functionality was measured by oposomal assay (OPA). New Zealand White rabbits were immunized intramuscularly with human doses (2.2 μg of polysaccharide, except for 6B, which was 4.4 μg) at weeks 0 and 2. Serum was sampled every 2 weeks after immunization.

[0315] Serotype-specific IgG concentration measurement Capsular polysaccharides (PnPs) of each serotype were coated onto 96-well plates at 0.5 μg / well to 1 μg / well. Equal amounts of serum were sampled from each subject and pooled by group. The serum pool was serially diluted 2.5-fold using antibody dilution buffer containing Tween 20 and pneumococcal cell wall polysaccharides (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 the absorbed, diluted serum was added to the coated well plate, followed by incubation at room temperature for 2 to 18 hours. The well plate was washed in the same manner, and then 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 above, and 1 mg / mL p-nitrophenylamine buffer was added to each well as a substrate, followed by incubation at room temperature for 2 hours. The reaction was quenched by adding 50 μl of 3 M NaOH and the absorbance was measured at 405 nm and 690 nm. The results are shown in Table 21.

[0316] [Table 21]

[0317] Functional immunogenicity assay (MOPA) Antibody function was assessed by testing sera in the MOPA assay. Pneumococcal MOPA strains stored at -70°C or below were diluted to the corresponding final dilution factor so that each strain had a concentration of approximately 50,000 CFU / mL. Equal amounts of serum were sampled from each subject, pooled by group, and serially diluted two-fold to leave 20 μl of serum in a U-bottom plate. After diluting the samples, 10 μl of the strain produced for each serotype was mixed with the diluted sample, and the mixture was incubated at room temperature for 30 minutes to ensure adequate mixing of the pneumococci and antibodies. A mixture of predifferentiated HL-60 cells and complement was added and 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 an agar plate that had been pre-dried for 30–60 minutes. The plate was then allowed to absorb for 20 minutes until dry. A 25 mg / mL TTC stock solution was added to the prepared overlay agar, and an appropriate antibody for the corresponding strain was added to it. 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 plate was incubated in a CO2 incubator (37°C) for 12–18 hours, after which colonies were counted. The MOPA titer was expressed as the dilution at which 50% killing was observed. The results are shown in Table 22.

[0318] [Table 22] [Example]

[0319] Formulation of a 27-valent pneumococcal conjugate vaccine with polysaccharides from serotypes 1, 5, 15B, and 22F conjugated to tetanus toxoid The monoconjugate was obtained according to the general method described in Examples 2-8. The desired volume of the final bulk concentrate was calculated based on the batch volume and bulk sugar concentration. 0.85% sodium chloride (saline), polysorbate 80, and succinate buffer were added to a pre-labeled formulation container, followed by the bulk concentrate. The product was then thoroughly mixed. The formulation was 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 if necessary. The formulated bulk product was stored at 2-8°C. The following multivalent pneumococcal conjugate vaccine formulation was manufactured and designated PCV27-(1 / 5 / 15B / 22F)-TT:

[0320] PCV27(1 / 5 / 15B / 22F)-TT contains polysaccharides of serotypes 1, 5, 15B, and 22F in TT and polysaccharides of serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 23A, 23B, 23F, 24F, 33F, and 35B in CRM. 197 and polysaccharide conjugates prepared by conjugating to a

[0321] PCV27(1 / 5 / 15B / 22F)-TT in a total dose of 0.5 ml contains 2.2 μg of each saccharide except for serotype 6B, which contains 4.4 μg, approximately 2 μg to 25 μg of TT (for serotypes 1, 5, 15B, and 22F), and approximately 45 μg to 100 μg of CRM. 197 , 0.125 mg of elemental aluminum (0.5 mg aluminum phosphate) adjuvant, 4.25 mg of sodium chloride, approximately 295 μg of succinate buffer solution, and approximately 120 μg of polysorbate 80. [Example]

[0322] Immunogenicity of the polyvalent pneumococcal conjugate vaccine, PCV27(1 / 5 / 15B / 22F)-TT The composite carrier polyvalent pneumococcal vaccine, PCV27(1 / 5 / 15B / 22F)-TT, prepared in Example 12, was tested for its ability to induce an immunogenic response in rabbits. Immunogenicity assessment was performed by antigen-specific ELISA for serum IgG concentration and by oposomosis assay (OPA) for antibody functionality. New Zealand White rabbits were immunized intramuscularly at weeks 0 and 2 with a human dose (2.2 μg of polysaccharide, except for 4.4 μg of 6B). Serum was sampled every two weeks after immunization. Serum was sampled every two weeks after immunization.

[0323] Serotype-specific IgG concentration measurement Capsular polysaccharides (PnPs) of each serotype were coated onto 96-well plates at 0.5 μg / well to 1 μg / well. Equal amounts of serum were sampled from each subject and pooled by group. The serum pool was serially diluted 2.5-fold using antibody dilution buffer containing Tween 20 and pneumococcal cell wall polysaccharides (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 the absorbed, diluted serum was added to the coated well plate, followed by incubation at room temperature for 2 to 18 hours. The well plate was washed in the same manner, and then 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 above, and 1 mg / mL p-nitrophenylamine buffer was added to each well as a substrate, 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 23.

[0324] [Table 23]

[0325] Functional immunogenicity assay (MOPA) When capsular polysaccharides of serotypes 1 and 5 are conjugated to TT, serotype-specific IgG concentrations increase when they are conjugated to CRMs. 197 Rabbits immunized with PCV27(1 / 5 / 15B / 22F)-TT also showed significantly increased IgG concentrations against 14 additional serotypes not present in PREVNAR13 (i.e., 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 22F, 23A, 23B, 24F, 33F, and 35B). In particular, serotypes 8 and 9N increased serum-specific IgG concentrations by more than 50-fold relative to PREVNAR13.

[0326] Antibody function was assessed by testing serum in the MOPA assay. Pneumococcal MOPA strains stored at -70°C or below were diluted to the corresponding final dilution factor so that each strain had a concentration of approximately 50,000 CFU / mL. Equal amounts of serum were sampled from each subject, pooled by group, and serially diluted two-fold to leave 20 μl of serum in a U-bottom plate. After diluting the sample, 10 μl of the strain prepared for each serotype was mixed with the diluted sample. The mixture was incubated at room temperature for 30 minutes to ensure sufficient mixing of the pneumococci and antibodies. A mixture of pre-differentiated 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 a pre-dried agar plate for 30-60 minutes, followed by drying. The plates were allowed to absorb for 20 minutes until the antibody was absorbed. A 25 mg / mL TTC stock solution was added to the prepared overlay agar, and the appropriate antibody for the corresponding strain was added to it. The mixture was mixed thoroughly, 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, and then the 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 24.

[0327] [Table 24]

[0328] When serotypes 1 and 5 are conjugated to TT, functional MOPA titers are higher than those of CRMs. 197Rabbits immunized with PCV27(1 / 5 / 15B / 22F)-TT also showed significantly increased functional MOPA titers against each of 14 additional serotypes not present in PREVNAR13 (i.e., 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 22F, 23A, 23B, 24F, 33F, and 35B).

[0329] While one or more exemplary embodiments have been described herein, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the inventive concept as defined by the following claims.

Claims

1. 1. A multivalent pneumococcal conjugate composition comprising 22 to 27 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, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B.

2. 2. The multivalent pneumococcal conjugate composition of claim 1, comprising 27 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, 15A, 15B, 15C, 18C, 19A, 19F, 23A, 23B, 22F, 23F, 24F, 33F, and 35B.

3. 2. The multivalent pneumococcal conjugate composition of claim 1, comprising 26 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, 33F, and 35B, and four serotypes selected from 15A, 15C, 23A, 23B, and 24F.

4. 2. The multivalent pneumococcal conjugate composition of claim 1, comprising 25 different pneumococcal capsular polysaccharide-protein conjugates, each pneumococcal capsular polysaccharide-protein conjugate comprising a protein carrier conjugated to 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, 33F, and 35B, and three serotypes selected from 15A, 15C, 23A, 23B, and 24F.

5. 2. The multivalent pneumococcal conjugate composition of claim 1, comprising 24 different pneumococcal capsular polysaccharide-protein conjugates, each pneumococcal capsular polysaccharide-protein conjugate comprising a protein carrier conjugated to 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, 33F, and 35B, and two serotypes selected from 15A, 15C, 23A, 23B, and 24F.

6. 2. The multivalent pneumococcal conjugate composition of claim 1, comprising 23 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 one serotype selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and 35B, and 15A, 15C, 23A, 23B, and 24F.

7. 2. The multivalent pneumococcal conjugate composition of claim 1, comprising 22 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, 33F, and 35B.

8. Protein carrier is CRM １９７ and / or tetanus toxoid.

9. At least two of the capsular polysaccharides are conjugated to tetanus toxoid, and the remaining capsular polysaccharides are CRM. １９７ 9. The multivalent pneumococcal conjugate composition of claim 8, wherein the at least two capsular polysaccharides conjugated to tetanus toxoid are selected from the group consisting of serotypes 1, 3, 5, 15B, and 22F.

10. Capsular polysaccharides from serotypes 1 and 5 were conjugated to tetanus toxoid, and capsular polysaccharides from serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B were conjugated to CRM. １９７ 3. The multivalent pneumococcal conjugate composition of claim 2, wherein the multivalent pneumococcal conjugate composition is conjugated to

11. Capsular polysaccharides from serotypes 1 and 3 were conjugated to tetanus toxoid, and capsular polysaccharides from serotypes 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B were conjugated to CRM. １９７ 3. The multivalent pneumococcal conjugate composition of claim 2, wherein the multivalent pneumococcal conjugate composition is conjugated to

12. Capsular polysaccharides from serotypes 3 and 5 were conjugated to tetanus toxoid, and capsular polysaccharides from serotypes 1, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B were conjugated to CRM. １９７ 3. The multivalent pneumococcal conjugate composition of claim 2, wherein the multivalent pneumococcal conjugate composition is conjugated to

13. Capsular polysaccharides from serotypes 1, 5, 15B, and 22F were conjugated to tetanus toxoid, and capsular polysaccharides from serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 23A, 23B, 23F, 24F, 33F, and 35B were conjugated to CRM. １９７ 3. The multivalent pneumococcal conjugate composition of claim 2, wherein the multivalent pneumococcal conjugate composition is conjugated to

14. Capsular polysaccharides from serotypes 1, 3, 15B, and 22F were conjugated to tetanus toxoid, and capsular polysaccharides from serotypes 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 23A, 23B, 23F, 24F, 33F, and 35B were conjugated to CRM. １９７ 3. The multivalent pneumococcal conjugate composition of claim 2, wherein the multivalent pneumococcal conjugate composition is conjugated to

15. Capsular polysaccharides from serotypes 3, 5, 15B, and 22F were conjugated to tetanus toxoid, and capsular polysaccharides from serotypes 1, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 23A, 23B, 23F, 24F, 33F, and 35B were conjugated to CRM. １９７ Conjugated to The multivalent pneumococcal conjugate composition of claim 2 .

16. The multivalent pneumococcal conjugate composition of any one of claims 1 to 15, further comprising an adjuvant.

17. 17. The multivalent pneumococcal conjugate composition of claim 16, wherein the adjuvant is an aluminum-based adjuvant.

18. 18. The multivalent pneumococcal conjugate composition of claim 17, wherein the adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide.

19. 20. The multivalent pneumococcal conjugate composition of claim 18, wherein the adjuvant is aluminum phosphate.

20. 20. Use of the multivalent pneumococcal conjugate composition of any one of claims 1 to 19 for the prevention of Streptococcus pneumoniae infection or disease in a subject.

21. A vaccine comprising the multivalent pneumococcal conjugate composition of any one of claims 1 to 19 and a pharmaceutically acceptable excipient.

22. 22. A method of preventing pneumococcal infection or disease in a subject, the method comprising administering to the subject a prophylactically effective amount of a multivalent pneumococcal conjugate composition of any one of claims 1 to 19 or a vaccine of claim 21.

23. 23. The method of claim 22, wherein the subject is a human who is at least 50 years of age and the disease is pneumonia or invasive pneumococcal disease (IPD).

24. 23. The method of claim 22, wherein the subject is a human who is at least 6 weeks old and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM).

25. 25. The method of claim 24, wherein the subject is between 6 weeks and 5 years old, between 2 and 15 months old, or between 6 and 17 years old.

26. 23. The use according to claim 20 or the method according to claim 22, wherein the subject is a human.

27. The method of any one of claims 22 to 26, wherein the multivalent pneumococcal conjugate composition or vaccine is administered by intramuscular injection.

28. The method of any one of claims 22 to 27, wherein the multivalent pneumococcal conjugate composition or vaccine is administered as part of an immunization series.

29. 1. An immunogenic composition comprising at least one polysaccharide-protein conjugate, wherein the polysaccharide in the at least one polysaccharide-protein conjugate is a capsular polysaccharide from Streptococcus pneumoniae serotype 15A, serotype 15C, serotype 23A, serotype 23B, serotype 24F, or serotype 35B.

30. A method of producing capsular polysaccharides from Streptococcus pneumoniae serotype 15A, serotype 15C, serotype 23A, serotype 23B, serotype 24F, or serotype 35B as described herein.

31. the serotype is serotype 15A, and the method comprises: (i) subjecting purified S. pneumoniae serotype 15A polysaccharide to an acid hydrolysis reaction and heat or a microfluidizer, and then reacting with an oxidizing agent to produce activated S. pneumoniae serotype 15A polysaccharide; (ii) optionally lyophilizing the activated S. pneumoniae serotype 15A polysaccharide and carrier protein; (iii) suspending the activated S. pneumoniae serotype 15A polysaccharide and carrier protein in dimethyl sulfoxide (DMSO); (iv) reacting the activated S. pneumoniae serotype 15A polysaccharide and the carrier protein with a reducing agent to produce a S. pneumoniae serotype 15A polysaccharide-carrier protein conjugate; and (v) capping any unreacted aldehydes in the Streptococcus pneumoniae serotype 15A polysaccharide-carrier protein conjugate to produce an immunogenic conjugate comprising Streptococcus pneumoniae serotype 15A polysaccharide covalently linked to a carrier protein.

31. The method of claim 30, comprising:

32. the serotype is serotype 15C, and the method comprises: (i) reacting purified S. pneumoniae serotype 15C polysaccharide with an oxidizing agent to produce activated S. pneumoniae serotype 15C polysaccharide; (ii) optionally lyophilizing the activated S. pneumoniae serotype 15C polysaccharide and carrier protein; (iii) suspending the activated S. pneumoniae serotype 15C polysaccharide and carrier protein in dimethyl sulfoxide (DMSO) or phosphate buffer; (iv) reacting the mixture of activated serotype 15C polysaccharide and carrier protein with a reducing agent to produce a serotype 15C polysaccharide-carrier protein conjugate; and (v) capping any unreacted aldehydes in the serotype 15C polysaccharide-carrier protein conjugate to produce an immunogenic conjugate comprising Streptococcus pneumoniae serotype 15C polysaccharide covalently linked to a carrier protein.

31. The method of claim 30, comprising:

33. the serotype is serotype 23A, and the method comprises: (i) reacting purified S. pneumoniae serotype 23A with an oxidizing agent to produce activated S. pneumoniae serotype 23A polysaccharide; (ii) optionally lyophilizing the activated S. pneumoniae serotype 23A polysaccharide and carrier protein; (iii) suspending the activated S. pneumoniae serotype 23A polysaccharide and carrier protein in dimethyl sulfoxide (DMSO) or phosphate buffer; (iv) reacting the mixture of activated S. pneumoniae serotype 23A polysaccharide and carrier protein with a reducing agent to produce a S. pneumoniae serotype 23A polysaccharide-carrier protein conjugate; and (v) capping any unreacted aldehydes in the Streptococcus pneumoniae serotype 23A polysaccharide-carrier protein conjugate to produce an immunogenic conjugate comprising Streptococcus pneumoniae serotype 23A polysaccharide covalently linked to a carrier protein.

31. The method of claim 30, comprising:

34. the serotype is serotype 23B, and the method comprises: (i) reacting purified S. pneumoniae serotype 23B with an oxidizing agent to produce activated S. pneumoniae serotype 23B polysaccharide; (ii) optionally lyophilizing the activated S. pneumoniae serotype 23B polysaccharide and carrier protein; (iii) Activated Streptococcus pneumoniae serotype 23B polysaccharide and carrier protein in dimethyl sulfoxide. suspending in dimethyl sulfoxide (DMSO); (iv) reacting the mixture of activated S. pneumoniae serotype 23B polysaccharide and carrier protein with a reducing agent to produce a S. pneumoniae serotype 23B polysaccharide-carrier protein conjugate; and (v) capping any unreacted aldehydes in the Streptococcus pneumoniae serotype 23B polysaccharide-carrier protein conjugate to produce an immunogenic conjugate comprising Streptococcus pneumoniae serotype 23B polysaccharide covalently linked to a carrier protein.

31. The method of claim 30, comprising:

35. the serotype is serotype 24F, and the method comprises: (i) subjecting purified S. pneumoniae serotype 24F polysaccharide to an acid hydrolysis reaction or a microfluidizer, followed by reaction with an oxidizing agent to produce activated S. pneumoniae serotype 24F polysaccharide; (ii) optionally lyophilizing the activated S. pneumoniae serotype 24F polysaccharide and the carrier protein; (iii) suspending the activated S. pneumoniae serotype 24F polysaccharide and carrier protein in dimethyl sulfoxide (DMSO) or phosphate buffer; (iv) reacting the activated S. pneumoniae serotype 24F polysaccharide and the carrier protein with a reducing agent to produce a S. pneumoniae serotype 24F polysaccharide-carrier protein conjugate; and (v) capping any unreacted aldehydes in the Streptococcus pneumoniae serotype 24F polysaccharide-carrier protein conjugate to produce an immunogenic conjugate comprising Streptococcus pneumoniae serotype 24F polysaccharide covalently linked to a carrier protein.

31. The method of claim 30, comprising:

36. the serotype is serotype 35B, and the method comprises: (i) reacting purified S. pneumoniae serotype 35B with an oxidizing agent to produce activated S. pneumoniae serotype 35B polysaccharide; (ii) optionally lyophilizing the activated S. pneumoniae serotype 35B polysaccharide and carrier protein; (iii) suspending the activated S. pneumoniae serotype 35B polysaccharide and carrier protein in dimethyl sulfoxide (DMSO) or phosphate buffer; (iv) reacting the activated S. pneumoniae serotype 35B polysaccharide and the carrier protein with a reducing agent to produce a S. pneumoniae serotype 35B polysaccharide-carrier protein conjugate; and (v) capping any unreacted aldehydes in the Streptococcus pneumoniae serotype 35B polysaccharide-carrier protein conjugate to produce an immunogenic conjugate comprising Streptococcus pneumoniae serotype 35B polysaccharide covalently linked to a carrier protein.

31. The method of claim 30, comprising:

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