EA044822B1 - POLYVALENT ANTI-NEUMOCOCCAL POLYSACCHARIDE-PROTEIN CONJUGED COMPOSITION - Google Patents

Description

Cross reference to related applications

This application claims priority and is based on the filing date of U.S. Provisional Patent Application No. 62/626,482 filed February 5, 2018 and Korean Patent Application No.

10-2018-0045246, filed April 18, 2018, the entire disclosure of which is incorporated herein by reference.

Field of technology

This application relates generally to multivalent anti-pneumococcal mixed-carrier conjugate compositions, vaccines containing them, and methods of using these compositions and vaccines to prevent a subject from Streptococcus pneumoniae infection or disease.

State of the art

Pneumococci (Streptococcus pneumoniae) are Gram-positive, spear-shaped, facultative anaerobic bacteria with more than 90 known serotypes. Most S. pneumoniae serotypes have been found to cause disease, with the 23 most common serotypes responsible for approximately 90% of invasive disease worldwide. Serotypes are classified based on the serological response to capsular polysaccharides, the most important virulence factor for pneumococci. Capsular polysaccharides are T cell-independent antigens that induce antibody production in the absence of T helper cells. T cell-independent antigens typically induce low-affinity antibodies and short-lived immune responses with little or no immunological memory.

The original pneumococcal vaccines included combinations of capsular polysaccharides from different serotypes. These vaccines can confer immunity against S. pneumoniae in patients with developed or healthy immune systems, but they are not effective in infants, who do not have a developed immune system, or in older adults, who often have compromised immune function. To improve the immune response to pneumococcal vaccines, especially in infants and the elderly who are at increased risk of developing S. pneumoniae infection, capsular polysaccharides have been conjugated to suitable carrier proteins to create pneumococcal conjugate vaccines. Conjugation to a suitable carrier protein changes the capsular polysaccharide from a T-cell independent antigen to a T-cell dependent antigen. Essentially, the immune response against the conjugated capsular polysaccharide involves T helper cells, which help induce a stronger and faster immune response upon re-exposure to the capsular polysaccharide.

There are at least two approaches to developing pneumococcal conjugate vaccines: the single-vehicle approach and the mixed-vehicle approach. The immunogenicity of conjugates of different capsular polysaccharides may differ depending on the pneumococcal serotype and the carrier protein used. In the single carrier approach, capsular polysaccharides from different serotypes are conjugated to a single protein carrier. Pfizer's PREVNAR vaccine series is an example of a single-carrier approach in which various capsular polysaccharides are conjugated to the protein carrier CRM197, a non-toxic variant of diphtheria toxoid having a single amino acid substitution from glutamic acid to glycine. The 7-valent PREVNAR vaccine (PREVNAR) was first approved in 2000 and contains capsular polysaccharides from the seven most common serotypes: 4, 6B, 9V, 14, 18C, 19F and 23F. The 13-valent vaccine, PREVNAR 13, adds serotypes 1, 5, 7F, 3, 6A and 19A to the CRM197 protein carrier. The protein carrier, CRM197, is a single carrier used in the PREVNAR vaccine and has never been used as part of a mixed-carrier system in the pneumococcal conjugate vaccine.

The second approach for pneumococcal vaccines is the mixed-carrier approach. In the mixed carrier approach, instead of using a single protein carrier, two or more protein carriers are used, with capsular polysaccharides from specific serotypes conjugated to a first protein carrier and capsular polysaccharides from other serotypes 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 the D H influenzae protein as protein carriers. , tetanus toxoid and diphtheria toxoid. In SYNFLORIX, serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F are conjugated to protein D; serotype 18C is conjugated to tetanus toxoid and serotype 19F is conjugated to diphtheria toxoid [2]. Serotype 3 was removed from the 11valent precursor SYNFLORIX, in part because it did not show serotype-specific efficacy in a study with acute otitis media [1]. Another group, Aventis Pasteur, developed an 11-valent (serotype 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F), mixed-carrier, pneumococcal conjugate vaccine using diphtheria toxoid as protein carriers and tetanus toxoid [3]. Capsular polysaccharides from serotypes 3.9V, 14 and 18C may produce a greater response when they are conjugated to diphtheria toxoid than when they are conjugated to tetanus toxoid [6]. Thus, serotypes 3, 6B, 14 and 18C were conjugated with diphtheria toxoid, and serotypes 1, 4, 5, 7F, 9V, 19F and 23F were conjugated with tetanus toxoid. Development of this mixed-carrier pneumococcal vaccine was discontinued, in part, for technical reasons and the potential for reduced response when administered with acellular pertussis vaccines [3]. It was recently reported that serotype 5, as well as 1, had one of the lowest observed OPA (opsonophagocytic antibody) titers of all PREVNAR 13 serotypes, in which an associated correlation was observed between IgG titer and OPA activity [4]. It has also been suggested that for serotype 3 a much higher serum IgG concentration would be required for protection [5].

The essence of the invention

This application provides new and improved multivalent mixed-carrier anti-pneumococcal conjugate compositions and vaccines containing them. In one aspect of the invention, this application provides a multivalent anti-pneumococcal mixed-carrier conjugate composition comprising 21 different pneumococcal capsular polysaccharide-protein conjugates, each pneumococcal capsular polysaccharide-protein conjugate containing a protein carrier conjugated to a capsular polysaccharide from a different Streptococcal serotype cus pneumoniae, while Streptococcus pneumoniae serotypes selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, the protein carrier being is CRM ₁₉₇ or tetanus toxoid, wherein two of the capsular polysaccharides are conjugated to tetanus toxoid and the remaining capsular polysaccharides are conjugated to CRM ₁₉₇ , and wherein the two capsular polysaccharides that are conjugated to tetanus toxoid are selected from the group consisting of serotypes 1, 3 and 5.

In some embodiments of the 21-valent mixed-carrier anti-pneumococcal conjugate composition, 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, 15B, 18C, 19A, 19F, 22f, 23F and 33F are conjugated to CRM ₁₉₇ .

In another embodiment of the 21-valent anti-pneumococcal mixed-carrier conjugate composition, 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, 15B, 18C, 19A, 19F, 22F, 23F and 33F are conjugated to CRM ₁₉₇ .

In another embodiment of the 21-valent anti-pneumococcal mixed-carrier conjugate composition, 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, 15B, 18C, 19A, 19F, 22F, 23F and 33F are conjugated to CRM ₁₉₇ .

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

Another aspect of the invention relates to the use of a 21-valent mixed-carrier anti-pneumococcal conjugate composition as a vaccine.

Another aspect of the invention relates to a vaccine containing a 21-valent anti-pneumococcal conjugate composition with a mixed carrier and a pharmaceutically acceptable excipient.

Another aspect relates to a method of preventing Streptococcus pneumoniae infection or disease in a subject such as a human, the method comprising administering to the subject a prophylactically effective amount of 21-valent anti-pneumococcal mixed-carrier conjugate compositions or a vaccine containing them.

In some embodiments, the subject is a person at least 50 years of age and the disease is pneumonia or invasive pneumococcal disease (IPD).

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

In some embodiments, the 21-valent anti-pneumococcal mixed-carrier conjugate composition or vaccine is administered by intramuscular injection.

In some embodiments, the 21-valent anti-pneumococcal mixed-carrier conjugate composition or vaccine is administered as part of an immunization series.

Another aspect relates to an immunogenic conjugate of Streptococcus pneumoniae serotype 9N, which contains: Streptococcus pneumoniae serotype 9N capsular saccharide; and a carrier protein associated with the capsular saccharide, wherein the carrier protein is CRM ₁₉₇ . In some embodiments, multivalent anti-pneumococcal conjugate compositions and vaccines with

- 2 044822 immunogenic serotype 9N and mixed carrier conjugate (and methods/uses thereof), the serotype 9N saccharide can be coupled to CRM197 to form the conjugate in a state in which it is activated to produce an oxidation state of 2-19 or 5-10 and molecular weight 200-700 kDa. In some embodiments of multivalent anti-pneumococcal serotype 9N conjugate and mixed-carrier conjugate compositions and vaccines (and methods/uses thereof), the immunogenic serotype 9N conjugate may have a molecular weight of 500-4000 kDa. In some embodiments of multivalent anti-pneumococcal serotype 9N conjugate and mixed-carrier conjugate compositions and vaccines (and methods/uses thereof), the ratio of serotype 9N capsular saccharide to carrier protein in the serotype 9N immunogenic conjugate is 0.1-5 ( w/w). In some embodiments, the ratio is 0.5-2.5.

In some embodiments of multivalent anti-pneumococcal serotype 9N conjugate and mixed-carrier conjugate compositions and vaccines (and methods/uses thereof), 15-60% of the 9N immunogenic conjugate may have a K _d value of 0 on the CL-4B column. 3 or lower.

In some embodiments of multivalent anti-pneumococcal serotype 9N conjugate and mixed-carrier conjugate compositions and vaccines (and methods/uses thereof), the serotype 9N immunogenic conjugate can be formulated with a serotype 9N polysaccharide that is activated to produce an oxidation state of 2-19 . In some embodiments, an immunogenic serotype 9N conjugate can be prepared with a serotype 9N polysaccharide that is activated to produce an oxidation state of 5-10.

In some embodiments of multivalent anti-pneumococcal conjugate compositions and vaccines with an immunogenic serotype 9N conjugate and a mixed carrier (and methods/uses thereof), if the serotype 9N Streptococcus pneumoniae saccharide is conjugated to CRM ₁₉₇ by adding 0.02-0.19 μg of periodate per 1 μg of sugar, then the conjugate can have a molecular weight of 500-4000 kDa, a molecular weight distribution of 15-60% (K _d < 0.3) and a saccharide/protein ratio of 0.5-2.5. In yet another aspect, the present disclosure also provides a method for preparing an immunogenic conjugate of Streptococcus pneumoniae serotype 9N, the method comprising:

(a) lysing a bacterial cell producing the capsular polysaccharide of Streptococcus pneumoniae serotype 9N by treating it with enzymes;

(b) purifying Streptococcus pneumoniae serotype 9N capsular saccharide from the lysed cell;

(c) activating the Streptococcus pneumoniae serotype 9N capsular polysaccharide by reacting with an oxidizing agent to produce an oxidation state of 2-19 or 5-10; and (d) forming a conjugate of the Streptococcus pneumoniae serotype 9N capsular saccharide bound to CRM ₁₉₇ by mixing the activated saccharide with CRM ₁₉₇ .

In some embodiments, CRM ₁₉₇ mixed in step (d) may react with a reducing agent to form a conjugate with an activated capsular polysaccharide of Streptococcus pneumoniae serotype 9N. In some embodiments, in step (c), 0.02-0.19 μg of periodate can react with 1 μg of Streptococcus pneumoniae serotype 9N capsular polysaccharide at 20-25°C for 15-20 hours.

In some embodiments, the Streptococcus pneumoniae serotype 9N capsular polysaccharide reacted with the oxidizing agent in step (c) may have a molecular weight of 400-900 kDa. In some embodiments, the activated capsular polysaccharide of Streptococcus pneumoniae serotype 9N mixed with CRM ₁₉₇ in step (d) may have a molecular weight of 200-700 kDa. In some embodiments, the immunogenic conjugate of Streptococcus pneumoniae serotype 9N may have a molecular weight of 500-4000 kDa. In some embodiments, the initial starting ratio of CRM ₁₉₇ to activated capsular saccharide serotype 9N (CRM ₁₉₇ carrier:saccharide) may be 0.5-2.5:1. In some embodiments, at least 15-60% of the immunogenic conjugate may have a Kd value of 0.3 or lower, measured on a CL-4B column.

In some embodiments, the Streptococcus pneumoniae serotype 9N polysaccharide described herein is conjugated to CRM ₁₉₇ by adding 0.02-0.19 μg of periodate per 1 μg of sugar, the immunogenic conjugate has a molecular weight of 500-4000 kDa, molecular weight distribution of 15-60 % ( _Kd x 0.3) as measured on a CL-4B column, and a CRM ₁₉₇ /polysaccharide ratio of 0.5-2.5.

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

Definitions

To make the present disclosure clearer, certain terms are first defined below. Additional definitions of the following terms and other terms may be set forth in

- 3 044822 description.

When used in this specification and the accompanying claims, the singular nouns include references to the plural unless the context clearly indicates otherwise.

Thus, for example, reference to a method includes one or more methods and/or steps of the type described herein and/or that will become apparent to those skilled in the art upon reading this disclosure, etc.

Administer: As used herein, to administer a composition to a subject means to give, apply, or bring the composition into contact with the subject. Administration can be by any of a number of routes, such as topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal, and intradermal.

Approximately: As used herein, the term approximately or about, as applied to one or more values of interest, refers to a value that is close to the specified nominal value. In some embodiments, the term about or about refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (more or less) of the stated nominal value unless stated otherwise or otherwise not obvious from the context (unless such a number would exceed 100% of the possible value).

Conjugate:

As used herein and understood from the relevant context, the terms conjugate(s) or glycoconjugate(s) refer to a Streptococcus pneumoniae polysaccharide conjugated to a carrier protein using any covalent or non-covalent bioconjugation strategy.

Oxidation state:

As used herein, the term degree of oxidation (DO) refers to the number of sugar repeat units per aldehyde group obtained when a purified or size-selected saccharide is activated by an oxidizing agent. The degree of oxidation of a saccharide can be determined using routine methods known to those of ordinary skill in the art.

Excipient:

As used herein, the term excipient refers to a non-therapeutic agent that can be included in a composition, for example, to provide or impart a desired consistency or stabilizing effect.

Mixed media:

As used herein, a mixed-carrier anti-pneumococcal conjugate composition refers to an anti-pneumococcal conjugate composition containing more than one type of protein carrier.

21-valent anti-pneumococcal mixed-carrier conjugate composition: As used herein, the term 21-valent anti-pneumococcal mixed-carrier conjugate composition(s) refers to a composition containing or consisting of 21 different pneumococcal capsular conjugates polysaccharides-proteins, wherein each pneumococcal capsular polysaccharide-protein conjugate contains a protein carrier conjugated to a capsular polysaccharide from various serotypes of Streptococcus pneumoniae, wherein the serotypes of Streptococcus pneumoniae are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein the protein carrier is CRM ₁₉₇ or tetanus toxoid, wherein two of the capsular polysaccharides are conjugated to tetanus toxoid and the remaining capsular polysaccharides conjugated to CRM ₁₉₇ , and wherein the two capsular polysaccharides that are conjugated to tetanus toxoid are selected from the group consisting of serotypes 1, 3, and 5. In some embodiments, the capsular polysaccharides from serotypes 1 and 5 are conjugated to tetanus toxoid, and the capsular polysaccharides from other serotypes are conjugated to CRM ₁₉₇ . In some embodiments, capsular polysaccharides from serotypes 1 and 3 are conjugated to tetanus toxoid, and capsular polysaccharides from the remaining serotypes are conjugated to CRM ₁₉₇ . In one embodiment, the capsular polysaccharides from serotypes 3 and 5 are conjugated to tetanus toxoid and the remaining capsular polysaccharides are conjugated to CRM ₁₉₇ .

Molecular mass:

Unless otherwise specified, as used herein, 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) in combination with multi-angle laser scattering (MALLS) analysis.

Polyvalent:

As used herein, the term multivalent refers to an anti-pneumococcal conjugate composition containing a pneumococcal capsular polysaccharide from more than

- 4 044822 one serotype of Streptococcus pneumoniae.

Pharmaceutically acceptable excipient:

Pharmaceutically acceptable excipients used in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, ^15th edition (1975), describes compositions and dosage forms suitable for the pharmaceutical delivery of one or more therapeutic compositions, including vaccines, and additional pharmaceutical agents. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene glycol, water, ethanol and the like. In general, the nature of the excipient will depend on the particular route of administration that is intended to be used. For example, parenteral dosage forms typically contain injectable liquids that include pharmaceutically and physiologically acceptable liquids as carriers, such as water, physiological saline, balanced salt solutions, buffers, aqueous dextrose, glycerol, and the like. For solid compositions (eg, powder, pill, tablet or capsule forms), common non-toxic solid excipients may include, for example, pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions contemplated for use may contain small amounts of non-toxic excipients such as wetting or emulsifying agents, a surfactant, preservatives, as well as pH buffering agents, etc., such as sodium acetate or sorbitan monolaurate.

Prophylactically effective amount:

As defined herein, the term prophylactically effective amount or prophylactically effective dose refers to the amount or dosage 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 infection pneumoniae

Prevention:

The term prophylaxis as used herein means preventing the onset of a disease, delaying its onset, and/or reducing the frequency and/or severity of one or more symptoms of a particular disease, disorder, or condition (eg, Streptococcus pneumoniae infection). In some embodiments, prevention is assessed on a population basis such that the agent is considered to provide prevention against a particular disease, disorder or condition if there is a statistically significant reduction in the development, frequency and/or intensity of one or more symptoms of the disease, disorder or condition in the population affected by the disease, disorder or condition.

Subject:

As used herein, the term subject means any mammal, including mice, rabbits and humans. In some 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

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

This application provides new and improved multivalent mixed-carrier anti-pneumococcal conjugate compositions and vaccines containing them. Because the protein carrier, CRM ₁₉₇ , has previously been used in single carrier pneumococcal conjugate vaccines, this application describes the use of CRM ₁₉₇ in a mixed carrier pneumococcal conjugate vaccine. In particular, this application describes the combined use of CRM ₁₉₇ and tetanus toxoid as carrier proteins for specific pneumococcal serotypes in multivalent anti-pneumococcal conjugate compositions and vaccines. As discussed above, the immunogenicity of conjugates of various capsular polysaccharides may differ depending on the pneumococcal serotype and the carrier protein used. This appendix describes the successful conjugation of serotype 3 to tetanus toxoid in a mixed-carrier vaccine, despite previous findings that serotype 3 was more immunogenic when conjugated to diphtheria toxoid rather than tetanus toxoid [6]. The application also describes the successful conjugation of serotypes 1 and 5 with tetanus toxoid in a mixed-carrier vaccine. It also discloses the unexpected discovery that antibody response to serotype 3 conjugated to tetanus toxoid in a multivalent, eg 21-valent, mixed-carrier anti-pneumococcal conjugate composition was approximately 4-5 times higher than when serotype 3 was conjugated to CRM ₁₉₇ in 13-valent anti-pneumococcal conjugate composition with a single carrier (PREVNAR 13).

- 5 044822

Moreover, this unexpected finding was not limited to serotype 3, but was also observed for other serotypes conjugated to tetanus toxoid in a multivalent mixed-carrier anti-pneumococcal conjugate composition. As shown in the examples, conjugation of serotypes 1 and 5 or 3 and 5 with tetanus toxoid with a mixed carrier, anti-pneumococcal conjugate composition with the remaining serotypes conjugated to CRM ₁₉₇ (for example, PCV21(l/5)-TT and PCV21(3/5) -TT), consistently induced significantly enhanced antibody responses to tetanus toxoid-conjugated serotypes compared with antibody responses (IgG responses or MOPA titers) against the same serotypes conjugated to CRM ₁₉₇ in an anti-pneumococcal conjugate formulation (PREVNAR 13) with a single vehicle . The 21-valent mixed-carrier pneumococcal conjugate composition described herein also includes pneumococcal serotypes not currently covered by the three pneumococcal conjugate vaccines currently available on the global market: PREVNAR (called Prevenar in some countries), SYNFLORIX and PREVNAR 13. The incidence of diseases caused by currently uncovered pneumococcal serotypes is increasing, partly due to the development of antibacterial resistance, an increase in the number of immunocompromised patients and lack of immune suppression. For example, none of the currently available pneumococcal conjugate vaccines contain serotype 9N. In addition, none of the currently available pneumococcal conjugate vaccines contain serotypes 8, 10A, 11A, 12F, 15B, 22F, and 33F. The present disclosure demonstrates the successful implementation of serotypes 8, 9N, 10A, 11A, 12F, 15B, 22F and 33F in a mixed-carrier pneumococcal conjugate vaccine (tetanus toxoid and CRM197), and that serotype 9N induced antibody responses that were about 40 to 50 times higher than antibody production from PREVNAR 13.

Pneumococcal polysaccharide serotype 9N

The serotype 9N polysaccharide can be obtained directly from bacteria using isolation procedures known to those of ordinary skill in the art (including, but not limited to, methods disclosed in US Patent Application Publication No. 2006/0228380). In addition, the saccharide can be produced using synthesis protocols.

Streptococcus pneumoniae serotype 9N can be obtained from standardized culture collections (eg, Centers for Disease Control and Prevention Streptococcal Reference Laboratory, Atlanta, Georgia) or clinical specimens. The bacterial cell is usually grown in a medium such as soybean medium. After fermentation of a bacterial cell producing capsular polysaccharide of Streptococcus pneumoniae serotype 9N, the bacterial cell is lysed to obtain a cell lysate. The serotype 9N polysaccharide can be isolated from a cell lysate using purification techniques known in the art, including centrifugation, depth filtration, sedimentation, ultrafiltration, activated carbon treatment, diafiltration and/or column chromatography (including, but not limited to, the methods disclosed in the publication application US Patent No. 2006/0228380). Purified serotype 9N capsular polysaccharide can be used to prepare an immunogenic conjugate. The serotype 9N capsular polysaccharide obtained by purifying the serotype 9N polysaccharide from a Streptococcus pneumoniae lysate, and the optionally size-selected purified polysaccharide, can be characterized by various parameters, including, for example, the molecular weight (MW) of the serotype 9N capsular polysaccharide. In some embodiments, the purified polysaccharide purified from Streptococcus pneumoniae serotype 9N prior to conjugation may have a molecular weight of 5-5000 kDa. In some embodiments, the serotype 9N capsular polysaccharide may have a molecular weight of 50-1000 kDa before conjugation. In some embodiments, the serotype 9N capsular polysaccharide may have a molecular weight of 70-900 kDa before conjugation. In some embodiments, the serotype 9N capsular polysaccharide may have a molecular weight of 100-800 kDa before conjugation. In some embodiments, the purified serotype 9N capsular polysaccharide can be activated prior to conjugation to produce a molecular weight of 50-800 kDa, 80-780 kDa, 100-770 kDa, 120-760 kDa, 140-750 kDa, 150-740 kDa, 160 -730 kDa, 170-735 kDa, 180-720 kDa, 190-710 kDa, 200-700 kDa, 220-690 kDa, 240-680 kDa, 260-670 kDa, 270-660 kDa or similar molecular weight ranges. Any integer within any of the above ranges is contemplated as an embodiment of the invention as described herein. Activated polysaccharide serotype 9N can be characterized by oxidation state and molecular weight. In some embodiments, the serotype 9N activated polysaccharide may have an oxidation state of 0.5-25; 0.6-23; 0.8-21; 1-20.8; 1.1-20.5; 1.2-20.3; 1.3-20; 1.4-19.5; 1.5-19.3; 1.6-19.2; 1.7-19.1; 2-19, 3-18, 4-15 or 5-10.

The polysaccharide may become slightly smaller in size during normal purification procedures. In addition, as described herein, the polysaccharide can be subjected to size selection prior to conjugation. The molecular weight range mentioned above applies to such purified polysaccharide after a final size selection step (eg, purification, hydrolysis and activation) prior to conjugation.

Polyvalent anti-pneumococcal conjugate compositions with mixed carriers and methods for their preparation

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

In some embodiments, 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 one embodiment, the capsular polysaccharides from serotypes 1 and 3 are conjugated to tetanus toxoid and the remaining capsular polysaccharides are conjugated to CRM ₁₉₇ . In one embodiment, the capsular polysaccharides from serotypes 3 and 5 are conjugated to tetanus toxoid and the remaining capsular polysaccharides are conjugated to CRM ₁₉₇ .

In a polysaccharide-protein conjugate vaccine, a carrier protein is conjugated to a polysaccharide antigen primarily to promote an immune response (eg, antibody production) to the polysaccharide antigen. The carrier proteins are preferably proteins that are non-toxic. The carrier proteins must be amenable to conjugation to the pneumococcal polysaccharide using standard conjugation procedures, as discussed in more detail below. The carrier proteins used in 21-valent mixed-carrier pneumococcal conjugate formulations are tetanus toxoid (TT) and CRM ₁₉₇ , each of which has been used in pneumococcal conjugate vaccines, but never in the same mixed-carrier vaccine.

CRM ₁₉₇ is a non-toxic variant (ie, toxoid) of diphtheria toxin that retains the immunological properties of wild-type diphtheria toxin. CRM ₁₉₇ differs from wild-type diphtheria toxin by one base in the structural gene, resulting in a single amino acid substitution from glutamic acid to glycine. CRM ₁₉₇ is usually isolated from cultures of the C7 (β 197) Corynebacterium diphtheria strain grown on a medium based on casein amino acids and yeast extract. CRM ₁₉₇ can be purified by ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography. Alternatively, CRM ₁₉₇ can be produced recombinantly in accordance with US patent No. 5614382, which is hereby incorporated by reference in its entirety. CRM ₁₉₇ has been used in pneumococcal conjugate vaccines, but never as a component of a mixed-carrier vaccine.

Tetanus toxoid is produced and used throughout the world for large-scale immunization against tetanus (or lockjaw) caused by Clostridium tetani. Tetanus toxoid is also used either alone or in combination with diphtheria and/or pertussis vaccines. The parent protein, tetanus toxin, is usually obtained from cultures of Clostridium tetani. Tetanus toxin is a protein weighing about 150 kDa and consists of two subunits (about 100 kDa and about 50 kDa) linked by a disulfide bond. The toxin is typically detoxicated with formaldehyde and can be purified from culture filtrates using known methods such as ammonium sulfate precipitation (see, for example, [7], [8]) or chromatographic techniques as described eg in WO 1996/025425. Tetanus toxin can also be inactivated by recombinant genetic agents.

Tetanus toxoid has also been used as a carrier protein in other vaccines, including pneumococcal conjugate vaccines. But the use of tetanus toxin in a mixed-carrier pneumococcal conjugate vaccine in combination with CRM ₁₉₇ represents a new approach. The prior art rejects the idea of conjugating serotype 3 to tetanus toxoid in a mixed-carrier pneumococcal conjugate vaccine because serotype 3 has been shown to be more immunogenic when conjugated to diphtheria toxoid compared to tetanus toxoid [6].

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, 15B, 18C, 19A, 19f, 22F, 23F and 33F, can be obtained from Streptococcus pneumoniae using any available technique, including standard techniques known to one of ordinary skill in the art, including, for example, those described in WO 2006/110381, WO 2008 /118752, WO 2006/110352, and publ. US patent applications No. 2006/0228380, 2006/0228381,

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2007/0184071, 2007/0184072, 2007/0231340, 2008/0102498 and 2008/0286838, all of which are incorporated herein by reference in their entirety. For example, each serotype of pneumococcal capsular polysaccharide can be grown in a culture medium (eg, soybean medium). The cells are lysed, and individual polysaccharides can be purified from the lysate by centrifugation, sedimentation, ultrafiltration and/or column chromatography. Additionally, pneumococcal capsular polysaccharide can be produced using synthesis protocols.

The capsular polysaccharide of Streptococcus pneumoniae contains repeating oligosaccharide units that can contain up to 8 sugar residues. The capsular saccharide antigen may be a full-length polysaccharide, or it may be reduced in size (eg, one oligosaccharide unit or shorter than the native length saccharide chain of repeating oligosaccharide units). The size of capsular polysaccharides can be reduced by various methods known in the art, such as acid hydrolysis treatment, hydrogen peroxide treatment, resizing with a high pressure homogenizer, followed by hydrogen peroxide treatment to produce oligosaccharide moieties, or microfluidization.

A pneumococcal conjugate of each serotype can be prepared by conjugating a capsular polysaccharide from each serotype to a carrier protein. Various pneumococcal conjugates can be prepared into a dosage form as a composition, including a single dose dosage form. To prepare a polysaccharide-protein conjugate, the capsular polysaccharide obtained from each pneumococcal serotype can be chemically activated so that the capsular polysaccharide can react with the carrier protein. Immediately after activation, each capsular polysaccharide can be separately conjugated to a carrier protein to form a glycoconjugate. Chemical activation of the polysaccharide and subsequent conjugation with a carrier protein can be achieved by traditional methods.

For example, vicinal hydroxyl groups at the end of the capsular polysaccharide can be oxidized to aldehyde groups by oxidizing agents such as periodates (including sodium periodate, potassium periodate or periodic acid), as described, for example, in US Pat. US No. 4365170, 4673574 and 4902506, which are hereby incorporated by reference in their entirety. Periodate randomly oxidizes the vicinal hydroxyl groups of a carbohydrate to form a reactive aldehyde group and causes cleavage of the C-C bond. The term periodate includes both periodate and periodic acid. The term also includes both metaperiodate ( _{104_} ) and orthoperiodate ( _{1065_} ). The term periodate also includes various periodate salts, including sodium periodate and potassium periodate. In some embodiments, the polysaccharide can be oxidized in the presence of sodium metaperiodate.

In some embodiments, the periodate may be used in an amount of about 0.03-0.17 μg per 1 μg of polysaccharide. In some embodiments, the periodate may be used in an amount of about 0.025-0.18 μg or about 0.02-0.19 μg per 1 μg of polysaccharide. If desired, the saccharide can be activated within the above range. Outside this range, unsatisfactory results may occur.

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

For example, the spacer may be cystamine or cysteamine to produce a thiolated polysaccharide that can be coupled to the carrier through a thioether linkage obtained after reaction with a maleimide-activated carrier protein (for example, using N-[γ-maleimidobutyryloxy]succinimide ester (GMBS) )) or a halogenated acetylated carrier protein (e.g. using iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl(4-iodoacetyl)aminobenzoate (S1AB), sulfosuccinimidyl(4iodoacetyl)aminobenzoate (sulfo-SIAB), N- succinimidyl iodoacetate (SIA) or succinimidyl 3[bromoacetamido]propionate (SBAP)). Preferably, the cyanate ester (optionally prepared by COAP chemistry) is coupled to a hexanediamine or adipic acid dihydrazide (AOH) and the amino derivative of the saccharide is conjugated to a carrier protein using a carbodiimide chemistry technique (e.g., EDAC or EDC) via a carboxyl group on the protein carrier . Such conjugates are described, for example, in WO 93/15760, WO 95/08348 and WO 96/129094, all of which are hereby incorporated by reference in their entirety.

Conjugation of activated capsular polysaccharides and the carrier protein can be achieved, for example, by reductive amination, as described, for example, in publ. US patent applications Such conjugates are described, for example, in publ. No. 2006/0228380, 2007/0231340, 2007/0184071 and 2007/0184072, WO 2006/110381, WO 2008/079653 and WO 2008/143709, all of which are hereby incorporated herein by reference in their entirety. For example, the activated capsular polysaccharides and the carrier protein can react with the reducing agent to form a conjugate. Suitable reducing agents include borohydrides such as cyanobo

- 8 044822 sodium borohydride, borane pyridine, sodium triacetoxyborohydride, sodium or borohydride, or borohydride ion exchange resin. At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugates. Unreacted aldehyde groups can be blocked using a suitable blocking agent such as sodium borohydride (NaBH ₄ ). In one embodiment of the invention, the reduction reaction is performed in an aqueous solvent. In another embodiment of the invention, the reduction reaction is performed in an aprotic solvent. In one embodiment of the invention, the reduction reaction is performed in a solvent of DMSO (dimethyl sulfoxide) or DMF (dimethylformamide). Other possible reducing agents include, but are not limited to, amine-boranes such as pyridine-borane, 2-picoline-borane, 2,6diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3, or 5-ethyl -2-methylpyridineborane (PEMB).

The activated capsular polysaccharides can be conjugated directly to the carrier protein or indirectly through the use of a spacer or linker, such as a bifunctional linker. The linker is optionally heterobifunctional or homobifunctional, containing, for example, a reactive amino group and a reactive carboxylic acid group, 2 reactive amino groups or two reactive carboxylic acid groups. Other suitable conjugation techniques employ carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU, as described, for example, in International Patent Application Publication No. WO 98/ 42721, which is incorporated herein by reference in its entirety. Conjugation may involve a carbonyl linker, which can be formed by reacting the free hydroxyl group of the saccharide with 1,1'-carbonyldiimidazole (CD1) (see Bethell et al. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al. (1981) J. Chromatogr. 218:509-518) followed by reaction with the protein to form a carbamate bond. This process may involve reducing the anomeric end group 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.

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 by serotype. The ratio can be determined either by independent measurement of the amounts of protein and polysaccharide present, or by methods that give a direct ratio known in the art. Methods including 1H NMR spectroscopy or dual-tracking EC-HPLC-UV/Raman (e.g. refractive index and UV for total and protein content, respectively) can determine the saccharide/protein ratio profile from the size distribution of the conjugates, as well as by SEC -HPLC-MALLS or MALDI-TOF-MS.

The polysaccharide-protein conjugates thus obtained can be purified and enriched in various ways. These methods include concentration/diafiltration, column chromatography and depth filtration. The purified polysaccharide-protein conjugates are combined to produce a 21-valent mixed-carrier anti-pneumococcal conjugate formulation that can be used as a vaccine.

The preparation of the dosage form of the vaccine composition can be performed using methods recognized in the art. The vaccine composition is prepared in the form of a preparation, which must be compatible with the intended route of administration. Conjugates of individual pneumococcal capsular polysaccharide-proteins may be formulated together with a physiologically acceptable carrier to formulate the composition. Examples of such carriers include, but are not limited to, water, buffered saline, polyols (eg, glycerin, propylene glycol, liquid polyethylene glycol) and dextrose solution.

In some embodiments, the 21-valent mixed-carrier anti-pneumococcal conjugate composition further comprises an adjuvant. As used herein, an adjuvant refers to a substance or carrier that nonspecifically enhances the immune response to an antigen. Adjuvants may include a suspension of mineral substances (potassium alum, aluminum salts such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, aluminum hydroxyphosphate, etc.) on which the antigen is adsorbed; or a water-in-oil emulsion, in which the antigen solution is emulsified in mineral oil (eg, Freund's incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity. Immunostimulatory oligonucleotides (such as those comprising a CpG motif) can also be used as adjuvants (eg, see US Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705 and 6,429,199). Adjuvants also include biological molecules such as lipids and costimulatory molecules. Typical biological adjuvants include AS04 [9], IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL. In some embodiments, the adjuvant is an aluminum-based adjuvant. Typically, one dose of 0.5 ml of vaccine is prepared in the form of a preparation that contains

- 9 044822 about 0.1 mg to 2.5 mg of aluminum-based adjuvant. In other embodiments, one 0.5 mL dose of the vaccine is formulated into a formulation that contains between 0.1 to 2 mg, 0.1 to 1 mg, 0.1 to 0.5 mg, 0.1 to 0.2 mg, 0.125 to 2.5 mg, 0.125 to 0.5 mg, 0.125 to 0.2, or 0.125 to 0.25 mg aluminum-based adjuvant. In some embodiments, a single 0.5 mL dose of the vaccine is formulated that contains from about 0.125 to about 0.250 mg of aluminum-based adjuvant. In some embodiments, a single 0.5 mL dose of the vaccine is formulated that contains about 0.125 mg of an aluminum-based adjuvant. In some embodiments, a single 0.5 mL dose of the vaccine is formulated that contains about 0.250 mg of an aluminum-based adjuvant.

In specific embodiments of the invention, the adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide.

In certain embodiments of the invention, the adjuvant is aluminum phosphate. In some embodiments, the composition is for use as a vaccine against Streptococcus pneumoniae infection.

Characterization of pneumococcal capsular polysaccharide-protein carrier conjugates

In some embodiments, the polysaccharide-protein carrier conjugate may have a molecular weight of 100-10,000 kDa. In some embodiments, the conjugate has a molecular weight of 200-9000 kDa. In some embodiments, the conjugate has a molecular weight of 300-8000 kDa. In some embodiments, the conjugate has a molecular weight of 400-7000 kDa. In some embodiments, the conjugate has a molecular weight of 500-6000 kDa. In some embodiments, the conjugate has a molecular weight of 600-5000 kDa. In some embodiments, the conjugate has a molecular weight of 500-4000 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the invention as described herein. If the molecular weight is within the above range, the conjugate can be stably obtained in high yield. You can also reduce the proportion of free polysaccharide. In addition, increased immunogenicity can be obtained within the above molecular weight range. After purification of the individual polysaccharide-protein conjugates, they are formulated to formulate the immunogenic composition described herein.

The saccharide-protein conjugates of the serotypes herein can be characterized by the ratio of polysaccharide to protein carrier (amount of polysaccharide/amount of protein carrier, w/w).

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

If the ratio of polysaccharide to protein carrier is within the above range, the conjugate can be stably obtained in high yield. You can also reduce the proportion of free polysaccharide. In addition, increased immunogenicity can be obtained and the conjugate can remain stable without interference from other serotypes within the above range. The conjugates and immunogenic compositions herein may contain a free polysaccharide that is non-covalently conjugated to the protein carrier but is nonetheless present in the polysaccharide-protein carrier conjugate composition. The free polysaccharide may be non-covalently associated with the polysaccharide-protein carrier conjugate (ie, non-covalently bound to, adsorbed to, or entrapped in or by the polysaccharide-protein carrier conjugate). In some embodiments, the polysaccharide-protein carrier conjugate contains less than about 60%, about 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15% free polysaccharide of each serotype, based on the total amount of polysaccharide each serotype. In some embodiments, the polysaccharide-protein carrier conjugate of each serotype contains less than about 60% free polysaccharide of each serotype, based on the total amount of polysaccharide of each serotype. In some embodiments, the polysaccharide-protein carrier conjugate of each serotype contains less than about 50% free polysaccharide of each serotype, based on the total amount of polysaccharide of each serotype. In some embodiments, the polysaccharide-carrier protein conjugate of each serotype contains less than about 40% free polysaccharide of each serotype, based on the total amount of polysaccharide of each serotype. In some embodiments, the polysaccharide-carrier protein conjugate of each serotype contains less than about 30% free polysaccharide of each serotype, based on the total amount of polysaccharide of each serotype. In some embodiments, the polysaccharide-carrier protein conjugate of each serotype contains less than about 25% free polysaccharide of each serotype, based on the total amount of polysaccharide of each serotype. In some embodiments it is possible to

- 10 044822 of the invention, each serotype polysaccharide-protein carrier conjugate contains less than about 20% free polysaccharide of each serotype, based on the total amount of polysaccharide of each serotype. In some embodiments, the polysaccharide-carrier protein conjugate of each serotype contains less than about 15% free polysaccharide of each serotype, based on the total amount of polysaccharide of each serotype. In some embodiments, the polysaccharide-protein carrier conjugate of each serotype contains less than about 10% free polysaccharide of each serotype, based on the total amount of polysaccharide of each serotype. Each serotype polysaccharide-protein carrier conjugate can also be characterized by its molecular size distribution ( _Kd ). Size exclusion chromatography support (CL-4B; cross-linked agarose beads, 4%) can be used to determine the relative size distribution of the conjugate molecules. Size exclusion chromatography (SEC) is used in a gravity column to profile the size distribution of conjugate molecules by size. Large molecules are excluded from the pores of the carrier and elute faster than small molecules. A fraction collector is used to collect the column eluate. Fractions are checked by analysis for saccharide content using the colorimetric method. To determine Kd, the column is calibrated to establish the fraction in which molecules are completely excluded (V ₀ ; K = 0) and the fraction representing maximum retention ( Vi ; K d = 1). The fraction in which the specified sample attribute (V _e ) is achieved is correlated with Kd according to the equation Kd = (Ve-V0)/(Vi-V0).

In some embodiments, at least 15% of each serotype polysaccharide-protein carrier conjugate may have a Kd value of 0.3 or lower on the CL-4B column. In some embodiments, at least 20% of each serotype polysaccharide-protein carrier conjugate may have a K _d value of 0.3 or lower on the CL-4B column. In some embodiments, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85 % or 90% of each serotype polysaccharide-protein carrier conjugate may have a K _d value of 0.3 or lower on the CL-4B column. In some embodiments, at least 60% of each serotype polysaccharide-protein carrier conjugate may have a K _d value of 0.3 or lower on the CL-4B column. In some embodiments, at least 50-80% of each serotype polysaccharide-protein carrier conjugate may have a K _d value of 0.3 or lower on the CL-4B column. In some embodiments, at least 65-80% of each serotype polysaccharide-protein carrier conjugate may have a K _d value of 0.3 or lower on the CL-4B column. In some embodiments, at least 15-60% of each serotype saccharide-protein carrier conjugate may have a Kd value of 0.3 or lower on the CL-4B column.

Preventive methods and applications

In another aspect of the disclosure, a vaccine is provided comprising a 21-valent anti-pneumococcal conjugate composition with a mixed carrier and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient contains at least a buffer, such as a succinate buffer, a salt, such as sodium chloride, and/or a surfactant, such as a polyoxyethylene sorbitan ester (eg, polysorbate 80). In some embodiments, two polysaccharides selected from the group consisting of serotypes 1, 3, and 5 are conjugated with tetanus toxoid, and the remaining capsular polysaccharides are among 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F are conjugated to CRM ₁₉₇ (21-valent).

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

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

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

In some embodiments, the vaccine induces a protective immune response in a human subject against a disease caused by Streptococcus pneumoniae infection. In accordance with a further aspect, this disclosure provides a method for preventing Streptococcus pneumoniae infection or disease, the method comprising administering to a human subject a prophylactically effective amount of a 21-valent anti-pneumococcal mixed-carrier conjugate composition or a vaccine containing the same. The 21-valent anti-pneumococcal mixed-carrier conjugate composition or vaccine containing it can be administered by any route, including, for example, the systemic or mucosal route, as described in more detail below. In some embodiments, the human subject is an elderly person and the disease is pneumonia or invasive pneumococcal disease (IPD). In some embodiments, the elderly subject is at least 50 years of age. In another

- 11 044822 In other embodiments, the elderly subject is at least 55 years of age. In still other embodiments, the elderly subject is at least 60 years of age. In other embodiments, the human subject is a child and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM). In some embodiments of the invention, the child is 0-2 years old. In other embodiments of the invention, the child is between 2 and 15 months of age. In yet another embodiment, the human subject is between 6 weeks and 17 years of age and is a child and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM). In certain embodiments of the invention, the human subject is between 6 weeks and 5 years of age. In other embodiments, the human subject is between 5 and 17 years of age.

The amount of conjugate in each vaccine dose or prophylactically effective amount of the multivalent anti-pneumococcal mixed-carrier conjugate composition may be selected as an amount that provides prophylaxis without significant adverse effects. This amount may vary depending on the pneumococcal serotype. In general, each dose may include from about 0.1 μg to about 100 μg of polysaccharide, specifically from about 0.1 μg to about 10 μg, and more particularly from about 1 μg to about 5 μg. Optimal amounts of components for a particular vaccine can be determined by standard studies involving observation of appropriate immune responses in the subject. For example, the amount to vaccinate a human subject can be determined by extrapolation from animal studies. In addition, the dose can be determined empirically.

In some embodiments, the vaccine or 21-valent anti-pneumococcal mixed-carrier conjugate composition may be a single 0.5 mL dose formulated that contains from about 1 μg to about 5 μg of each capsular polysaccharide; from about 1 μg to about 30 μg TT; from about 20 μg to about 85 μg CRM ₁₉₇ ; and optionally from about 0.1 mg to about 0.5 mg of elemental aluminum adjuvant. In some embodiments, the vaccine or 21-valent anti-pneumococcal mixed-carrier conjugate composition may be a single 0.5 mL dose formulated into a formulation that contains from about 2 μg to about 2.5 μg of each capsular polysaccharide, excluding serotype 6B and optionally serotype 3; which is present in an amount of from about 4 μg to about 5 μg; about 2 μg to about 25 μg TT; about 40 μg to about 75 μg CRM ₁₉₇ ; and optionally from about 0.1 mg to about 0.25 mg of elemental aluminum adjuvant.

In some embodiments, the vaccine or 21-valent anti-pneumococcal mixed-carrier conjugate composition may be a single 0.5 mL dose formulated that contains about 2.2 μg of each capsular polysaccharide, excluding serotype 6B; which is present in an amount of about 4.4 μg. In some embodiments, the vaccine or 21-valent anti-pneumococcal mixed-carrier conjugate composition may be a single 0.5 mL dose formulated into a formulation that contains from about 2 μg to about 2.5 μg of each capsular polysaccharide, excluding up to six capsular polysaccharides selected from the group consisting of serotypes 1, 3, 4, 5, 6B, 9V, 19A and 19F; each of which is present in amounts from about 4 μg to about 5 μg. In one embodiment, 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 21-valent anti-pneumococcal mixed-carrier conjugate composition may be a single 0.5 mL dose formulated that contains about 2.2 μg of each capsular polysaccharide, excluding up to six capsular polysaccharides selected from the group consisting of serotypes 1, 3, 4, 5, 6B, 9V, 19A and 19F; each of which is present in amounts of about 4.4 mcg. In one embodiment of the invention, 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.

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

- 12 044822 about 2 to about 2.5 μg of capsular polysaccharides of serotypes 1, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F,

14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and from about 4 μg to about 5 μg of serotype 6B capsular polysaccharides, and from about 8 μg to about 9 μg of serotype 3 capsular polysaccharides, and more preferably about 8, 8 mcg capsular polysaccharide serotype 3.

In some embodiments, the 21-valent mixed-carrier anti-pneumococcal conjugate composition or vaccine containing the same further comprises a buffer of sodium chloride and sodium succinate as excipients.

In some embodiments, the 21-valent anti-pneumococcal mixed-carrier conjugate composition may be formulated in a liquid dosage form in which each of pneumococcal capsular polysaccharides of serotypes 1 and 3 is conjugated to TT, and capsular polysaccharides from serotypes 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F are conjugated to CRM ₁₉₇ (21-valent). Each 0.5 mL dose may be prepared as a liquid dosage form containing: about 2.2 μg of each capsular polysaccharide, except for serotype 6B at about 4.4 μg; from about 2 μg to about 25 μg of TT carrier protein (for serotypes 1 and 3 only) and from about 40 μg to about 75 μg of CRM carrier protein ₁₉₇ ; about 0.125 mg to about 0.250 mg elemental aluminum (about 0.5 to about 1.2 mg aluminum phosphate) as an adjuvant; and a buffer of sodium chloride and sodium succinate as excipients. In some embodiments, the 21-valent anti-pneumococcal mixed-carrier conjugate composition may be formulated in a liquid dosage form in which each of the pneumococcal capsular polysaccharides of serotypes 1 and 5 is conjugated to TT, and capsular polysaccharides from serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F are conjugated to CRM ₁₉₇ (21-valent). In one embodiment, each 0.5 mL dose can be formulated as a liquid dosage form containing: about 2.2 μg of each capsular polysaccharide, except for serotype 6B at about 4.4 μg and serotype 3 at about 2.2 -8.8 mcg; from about 2 μg to about 25 μg of TT carrier protein (for serotypes 1 and 5 only) and from about 40 μg to about 75 μg of CRM carrier protein ₁₉₇ ; about 0.125 mg to about 0.250 mg elemental aluminum adjuvant (about 0.5 to about 1.2 mg aluminum phosphate); and a buffer of sodium chloride and sodium succinate as excipients. In some embodiments, serotype 3 is present in an amount of about 2.2 μg. In other embodiments, serotype 3 is present in an amount of about 4.4 μg. In other embodiments, serotype 3 is present in an amount of about 8.8 μg. In yet another embodiment, each 0.5 mL dose may be formulated as a liquid dosage form containing: about 2.2 μg of each capsular polysaccharide, excluding up to six capsular serotypes selected from the group consisting of serotypes 1, 3, 4 , 5, 6B, 9V, 19A and 19F in an amount of about 4.4 μg; from about 2 μg to about 25 μg of TT carrier protein (for serotypes 1 and 5 only) and from about 40 μg to about 75 μg of CRM carrier protein ₁₉₇ ; about 0.125 mg to about 0.250 mg elemental aluminum adjuvant (about 0.5 to about 1.2 mg aluminum phosphate); and a buffer of sodium chloride and sodium succinate as excipients. In one embodiment, up to six capsular polysaccharides in an amount of about 4.4 μg are selected from the group consisting of serotypes 1, 3, 4, 6B, 9V, 19A and 19F. In another embodiment, each 0.5 ml dose can be formulated as a liquid dosage form containing: about 2.2 μg of each capsular polysaccharide, except for serotypes 3, 4, 6B, 9V, 19A and 19F in an amount of about 4.4 mcg; from about 2 μg to about 25 μg of TT carrier protein (for serotypes 1 and 5 only) and from about 40 μg to about 75 μg of CRM carrier protein ₁₉₇ ; about 0.125 mg to about 0.250 mg elemental aluminum adjuvant (about 0.5 to about 1.2 mg aluminum phosphate); and a buffer of sodium chloride and sodium succinate as excipients. In another embodiment, each 0.5 ml dose can be formulated as a liquid dosage form containing: about 2.2 μg of each capsular polysaccharide, excluding serotypes 3 and 4 in an amount of about 4.4 μg; from about 2 μg to about 25 μg of TT carrier protein (for serotypes 1 and 5 only) and from about 40 μg to about 75 μg of CRM carrier protein ₁₉₇ ; about 0.125 mg to about 0.250 mg elemental aluminum adjuvant (about 0.5 to about 1.2 mg aluminum phosphate); and a buffer of sodium chloride and sodium succinate as excipients.

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

In some embodiments, the liquid dosage form can be filled into single dose syringes without a preservative. After shaking, the liquid dosage form becomes a vaccine, which is a homogeneous white suspension, ready for intramuscular administration. The 21-valent anti-pneumococcal mixed-carrier conjugate composition can be administered as a single injection or as part of a series of immunizations. For example, a 21-valent mixed-carrier anti-pneumococcal conjugate composition can be administered 2, 3, 4 or more times at suitably spaced intervals, such as 1, 2, 3, 4, 5 or 6 month intervals, or a combination thereof. In some embodiments, the 21-valent anti-pneumococcal mixed-carrier conjugate composition is administered to the infant 4 times within the first 15 months after birth, including, for example, at about 2, 3, 4, and 12-15 months of age; at about 3, 4, 5 and 12-15 months of age; or at about 2, 4, 6 and 12-15 months of age. This first dose can be given as early as 6 weeks of age. In another embodiment of the invention, the 21-valent anti-pneumococcal conjugate composition with a mixed carrier is administered to the child 3 times within the first 15 months after birth, including, for example, about 2, 4 and 11-12 months. The multivalent anti-pneumococcal mixed-carrier conjugate composition may also include one or more proteins from Streptococcus pneumoniae. For example, 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. The 21valent anti-pneumococcal mixed-carrier conjugate composition may be administered to a subject via one or more routes of administration known to one of ordinary skill in the art, such as parenteral, transdermal or transmucosal, intranasal, intramuscular, intraperitoneal, intradermal, intravenous, or subcutaneous routes, and may be suitably formulated in the form of a drug. The 21-valent anti-pneumococcal mixed-carrier conjugate composition can be formulated into a formulation that is compatible with the intended route of administration.

In some embodiments, the 21-valent anti-pneumococcal mixed-carrier conjugate composition can be administered as a liquid dosage form by intramuscular, intraperitoneal, subcutaneous, intravenous, intra-arterial or transdermal injection, or respiratory mucosal injection. The 21-valent anti-pneumococcal mixed-carrier conjugate composition can be formulated in liquid form or lyophilized form. In some embodiments, the injectable compositions are prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. In some embodiments, injectable solutions and suspensions are prepared from sterile powders or granules. General recommendations for dosage formulation and production of pharmaceutical agents for administration by these routes can be found, for example, in Remington's Pharmaceutical Sciences, ^19th ed., Mack Publishing Co., Easton, PA, 1995; incorporated herein by reference. Currently, the oral or nasal spray or aerosol route (eg, inhalation) is most commonly used to deliver therapeutic agents to the lungs and respiratory system. In some embodiments, the 21-valent anti-pneumococcal conjugate composition with a mixed carrier is administered using a device that delivers a metered dosage of the composition. Suitable devices for use in the delivery of intradermal pharmaceutical compositions described herein include short needle devices such as those described in US Pat. No. 4,886,499, US Pat. No. 5,190,521, US Pat. No. 5,328,483, US Pat. No. 5,527,288, US Pat. US Patent No. 5,015,235, US Patent No. 5,141,496, US Patent No. 5,417,662 (all of which are incorporated herein by reference). Intradermal compositions can also be administered by devices that limit the effective penetration of a needle length into the skin, such as those described in WO 1999/34850, incorporated herein by reference, to their functional equivalents. Also suitable are jet injection devices that deliver liquid vaccines into the skin by means of a liquid jet injector or by means of a needle that pierces the stratum corneum of the skin and creates a jet that reaches the skin. Jet injection devices are described, for example, in US Patent No. 5480381, US Patent No. 5599302, US Patent No. 5334144, US Patent No. 5993412, US Patent No. 5649912, US Patent No. 5569189, US Patent No. 5704911, US Patent No. 5383851, US Patent No. US Patent No. 5893397, US Patent No. 5466220, US Patent No. 5339163, US Patent No. 5312335, US Patent No. 5503627, US Patent No. 5064413, US Patent No. 5520639, US Patent No. 4596556, US Patent No. 4790824, US Patent No. 49 41880, patent US No. 4940460, WO1997/37705 and WO1997/13537 (all of which are incorporated herein by reference). Ballistic powder/particle delivery devices are also useful, which use compressed gas to accelerate the vaccine in powder form to penetrate the outer layers of the skin into the epidermis. In addition, you can use ordinary syringes in the classic Mantoux method of intradermal administration.

Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene

- 14 044822 lenglycol, oils such as olive oil and injectable organic esters such as ethyl oleate. Examples of the oil include vegetable or animal oil, peanut oil, soybean oil, olive oil, sunflower oil, fish liver oil, synthetic oil such as marine oil, and lipids derived from milk or eggs. Aqueous media include water, alcohol/aqueous solution, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, dextrose Ringer's solution, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous carriers include fluid and nutrient replenishment solutions, electrolyte replenishment solutions (such as those based on dextrose Ringer's solution), etc. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents and inert gases and the like.

The 21-valent anti-pneumococcal mixed-carrier conjugate composition can be formulated in the form of a single dose vial, multiple dose vial or pre-filled syringe. A pharmaceutically acceptable carrier for a liquid dosage form includes an aqueous or non-aqueous solvent, suspension, emulsion or oil. The composition may be isotonic, hypertonic or hypotonic. However, it is desirable that the composition for infusion or injection be substantially isotonic. Since isotonicity or hypertonicity may provide an advantage for storing the composition. If the composition is hypertonic, it may be diluted before administration. The tonicity adjusting agent may be an ionic tonicity adjusting agent such as a salt or a non-ionic tonicity adjusting agent such as a carbohydrate. The tonicity adjusting agent includes, but is not limited to, sodium chloride, calcium chloride, potassium chloride and magnesium chloride. Non-ionic tonicity adjusting agent includes, but is not limited to, sorbitol and glycerol. Preferably included in at least one pharmaceutically acceptable buffer. For example, if the composition is an infusion or injection, it will preferably be prepared in a buffer with a buffer capacity at pH 4 to pH 10, such as pH 5 to pH 9, or pH 6 to pH 8. The buffer may be selected from corresponding to the United States Pharmacopoeia (USP). For example, the buffer may be selected from the group consisting of a monobasic acid such as acetic acid, benzoic acid, gluconic acid, glyceric acid and lactic acid; dibasic acid such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid and tartaric acid; polybasic acid such as citric acid and phosphoric acid; and bases such as ammonia, diethanolamine, glycine, triethanolamine and TRIS. The mixed-carrier 21-valent anti-pneumococcal conjugate composition may contain a surfactant. Examples of the surfactant include, but are not limited to, polyoxyethylene sorbitan ester (commonly referred to as Tween), particularly polysorbate 20 and polysorbate 80; copolymers (such as DOWFAX) of ethylene oxide (EO), propylene oxide (PO), butylene oxide (BO); octoxynols with various repeats of the ethoxy(hydroxy-1,2-ethanediyl) group, in particular octoxynol-9 (Triton-100); ethylphenoxypolyethoxyethanol (IGEPAL CA-630/NP-40); a phospholipid such as lecithin; nonylphenol ethoxylate, such as from the TERGITOL NP series; lauryl, cetyl, stearyl, oleyl fatty alcohol derivatives of polyoxyethylene ethers (Brij surfactant), in particular triethylene glycol monolauryl ether (Brij 30); sorbitan ester, known as SPAN, particularly sorbitan trioleate (Span 85) and sorbitan monolaurate.

Mixtures of surfactants such as Tween 80/Span 85 can be used. A combination of a polyoxyethylene sorbitan ester such as Tween 80 and an octoxynol such as Triton X-100 is also useful. The combination of Laureth 9 and Tween and/or octoxynol is advantageous. Preferably, the amount of polyoxyethylene sorbitan ester (such as Tween 80) included in the composition may be from 0.01 to 1% (w/v), from 0.01 to 0.1% (w/v), from 0 .01 to 0.05% (w/v) or about 0.02%; the amount of octylphenoxypolyoxyethanol or nonylphenoxypolyoxyethanol (such as Triton X-100) included in the composition may be from 0.001 to 0.1% (w/v), in particular from 0.005 to 0.02%; and the amount of polyoxyethylene ether (such as Laureth 9) included may be from 0.1 to 20% (w/v), possibly from 0.1 to 10%, in particular from 0.1 to 1% or about 0.5%.

In some embodiments of the invention, the 21-valent anti-pneumococcal conjugate composition with a mixed carrier can be delivered via a controlled release system. For example, intravenous infusion, transdermal patch, liposome, or other methods may be used for administration. In one aspect, macromolecules such as microspheres or an implant can be used.

The above description generally describes the present invention. A more complete understanding can be gained by referring to the following specific examples. These examples are described entirely for illustrative purposes and are not intended to limit the scope of the present invention.

Examples

Example 1. Preparation of capsular polysaccharides of S. pneumoniae

- 15 044822

Cultivation of S. pneumoniae and purification of capsular polysaccharides were carried out as known to anyone skilled in the art. Serotype S. pneumoniae was 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 6v: ATSS No. 6326; Serotype 7f: ACS No. 10351; Serotype 9n: ACS No. 6309; Serotype 9V: ACS No. 10368; Serotype 14: ACS No. 6314; Serotype 18s: ACS No. 10356; Serotype 19A: ACS No. 10357; Serotype 19f; Serotype 19F : ATCC No. 6319; serotype 23F: ATCC No. 6323). In-house strains were used for serotypes 8, 10A, 11A, 12F, 15B, 22F, and 33F, but any publicly available strain could be used. Characteristics of S. pneumoniae were determined by capsules and motility, gram-positive staining, spear-shaped diplococci, and alpha-hemolysis in blood agar medium.

Serotypes were identified by the Quelling test using specific antisera (US Pat. No. 5,847,112).

Obtaining cell banks

In order to propagate the strains and remove components of animal origin, several generations of seed cultures were obtained (generations F1, F2 and F3). Two additional generations of seed crops were obtained. The first additional generation was cultured from the F3 flask, and the subsequent generation was cultured from the first additional generation flask. Vials with seed cultures were stored frozen (below -70°C) with synthetic glycerol as a cryopreservative. To obtain a cell bank, all cultures were grown in soybean medium. Before freezing, cells were concentrated by centrifugation, depleted medium was removed, and pelleted cells were resuspended in fresh medium containing a cryopreservative (such as synthetic glycerol).

Cultivation and collection

Cultures from the working cell bank were inoculated into seed bottles containing soy medium and cultured. Once the target optical density (absorbance) was reached, the seed bottle was used to inoculate the fermenter containing soybean media. Cultivation was stopped when the optical density value began to remain constant. After cessation of culture, sodium deoxycholate was added to lyse the cells. The resulting fermenter contents were cooled and protein precipitation was initiated. The mixture was then centrifuged to remove precipitated proteins and cellular debris.

Cleaning

The solution obtained from centrifugation was filtered through a depth filter to remove proteins and cellular debris that did not settle during centrifugation. The filtrate was concentrated on a 100 kDa MW membrane and the concentrate was diafiltered with 10 volumes of 25 mM sodium phosphate buffer (pH 7.2) to obtain a sample. The sample was filtered to collect the supernatant, from which polysaccharides were precipitated and filtered. The filtrate was concentrated on a 30 kDa MW membrane and the concentrate was diafiltered using about 10 volumes of triple distilled water. After diafiltration was performed, the remaining solution was filtered through a 0.2 μm filter. In-process control testing was performed on the filtrate (appearance, residual proteins, residual nucleic acids, endotoxins, molecular weight values and total polysaccharides). The concentrate was sterile filtered and stored at -20°C.

Example 2. Preparation of a conjugate of S. pneumoniae capsular polysaccharide and carrier protein

Polysaccharides of different serotypes were activated following different pathways and then conjugated to the carrier protein CRM ₁₉₇ or TT. Specifically, the conjugates were prepared by conjugating each of the capsular polysaccharides of all serotypes to CRM ₁₉₇ and by conjugating each of the capsular polysaccharides of serotypes 1, 3 and 5 to TT. Depending on the size of the native serotype, the activation process may involve size reduction of each capsular polysaccharide to the target molecular weight, chemical activation, and buffer exchange via ultrafiltration. The conjugates were purified using ultrafiltration and ultimately filtered through a 0.2 μm filter. The process parameters such as pH, temperature, concentration and duration were as follows.

(1) Activation process

Stage 1. Hydrolysis

Reductive amination is a well-known method for conjugating polymers in which an amide bond is formed between the (-NH ₂ ) group of the primary amine of a protein and the aldehyde of a saccharide. Aldehyde groups are added to the pneumococcal capsular polysaccharide to create conditions for conjugation with the carrier protein. The vicinal diol structure of the monosaccharide can be oxidized with sodium periodate (NaIO ₄ ) to form aldehyde groups. Capsular polysaccharides from serotypes 1, 3, 4, 6A, 8, 11A, 12F, 14, 15B, 18C, 22F and 33F were processed as follows.

For serotype 1, sodium hydroxide was added to the capsular polysaccharide solution (to 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 thereto

- 16 044822 to a final pH value of 6.0±0.1, thereby stopping hydrolysis.

In the case of serotypes 3, 8, 11A and 15B, hydrochloric acid was added to the solution of the capsular polysaccharide (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 hydrolysis. In the case of 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. 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 a final pH of 6.0 ± 0.1, thereby stopping hydrolysis.

In the case of serotype 6A, glacial acetic acid was added to the solution of the 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 hydroxide was added to it to a final pH of 6.0 ± 0.1, thereby stopping hydrolysis.

In the case of serotype 12F, hydrochloric acid was added to the capsular polysaccharide solution (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 to the solution until the final pH of the solution was 6.0 ± 0.1, thereby stopping hydrolysis.

In the case of 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 hydrolysis. 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 hydrolysis.

Each of the resulting capsular polysaccharides was diluted in water for injection (WFI), sodium acetate and sodium phosphate were added to a final concentration of about 1.0 mg/ml and about 2.0 mg/ml.

Stage 2. Reaction with periodate

The molar equivalent of sodium periodate to activate each pneumococcal saccharide was determined based on the molar mass of the repeat unit. With thorough mixing, oxidation reactions were allowed to proceed for 16 to 20 hours at a temperature of 21 to 25°C for all serotypes except 1, 7F and 19F, for which the temperature was 10°C or less. To maintain constant and stable production of conjugates during the conjugation process, a target range of oxidation state (Do) levels has been established for each serotype. Preferably, the target range for Do levels for each serotype is shown in Table. 1 and 2.

Table 1. Do range for all serotypes that need to be conjugated to CRM ₁₉₇

Serotype Range Do Serotype Range Do Serotype 1 from 4 to 10 Serotype HA from 1 to 15 Serotype 3 from 2 to 8 Serotype 12F from 1 to 9 Serotype 4 from 1 to 5 Serotype 14 from 6 to 13 Serotype 5 from 2 to 6 Serotype 15B from 1 to 17 Serotype 6A from 5 to 15 Serotype 18C from 6 to 14 Serotype 6B from 7 to 13 Serotype 19A from 7 to 13 Serotype 7F from 2 to 8 Serotype 19F from 6 to 12 Serotype 8 from 1 to 17 Serotype 22F from 1 to 16 Serotype 9N from 5 to 10 Serotype 23F from 6 to 14 Serotype 9 from 4 to 9 Serotype 33F from 1 to 15 Serotype 10 A from 1 to 12

- 17 044822

Table 2. Do range for serotypes 1, 3 and 5 that need to be conjugated with TT

Serotype Range Do Serotype 1 (1-TT) from 1 to 15 Serotype 3 (3-TT) from 2 to 14 Serotype 5 (5-TT) from 1 to 15

Stage 3. Ultrafiltration

The oxidized saccharide was concentrated and diafiltered using WFI on a 100 kDa molecular weight cutoff (MWCO) ultrafilter (30 kDa ultrafilter for serotype 1 and 5 kDa ultrafilter for serotype 18C). Diafiltration was performed using 0.9% sodium chloride solution for serotype 1.0.01 M sodium acetate buffer (pH 4.5) for serotypes 7F and 23F and 0.01 M sodium phosphate buffer (pH 6.0) for serotype 19F . The permeate was discarded and the retenetate was filtered through a 0.2 μm filter.

Stage 4. Lyophilization

For capsular polysaccharides of serotypes 3, 4, 5, 8, 9N, 9V, 10A, 14, 15B, 22F and 33F, which need to be conjugated to the carrier protein using an aqueous solvent, a mixed solution of polysaccharides and carrier protein was prepared without adding additional sucrose , lyophilized and then stored at -25°C ± 5°C.

For capsular polysaccharides of serotypes 1 and 18C, which need to be conjugated to a protein carrier using an aqueous solvent, the polysaccharides and protein carrier were independently prepared without adding additional sucrose, lyophilized and then stored at -25°C ± 5°C. For capsular polysaccharides of serotypes 6A, 6B, 7F, 19A, 19F and 23F, which must be conjugated to the carrier protein using the solvent DMSO, a predetermined amount of sucrose was added to the activated polysaccharides to achieve a final sucrose concentration of 5% ± 3% (wt. vol.) and samples were independently prepared, lyophilized, and then stored at 25°C ± 5°C.

For serotype IA capsular polysaccharide, a predetermined amount of sucrose was added to the activated polysaccharide to achieve a final sucrose concentration of 20% ± 5% (w/v), and the polysaccharides and carrier protein were independently prepared, lyophilized, and then stored at -25°C. C ± 5°C.

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

(2) Conjugation process

Conjugation in aqueous medium was carried out for serotypes 1, 3, 4, 5, 8, 9N, 9V, 10A, 14, 15B, 18C, 22F and 33F, and conjugation in DMSO was carried out for serotypes 6A, 6B, 7F, 11 A, 12F , 19A, 19F and 23F. Each of the capsular polysaccharides was conjugated to a carrier protein in a ratio of 0.2 to 2:1.

Stage 1. Dissolution

Conjugation in aqueous media

For serotypes 1, 3, 4, 5, 8, 9N, 9V, 10A, 14, 15B, 18C, 22F, and 33F, the lyophilized sample was thawed and brought to room temperature. The lyophilized sample was dissolved to the reaction concentration by using a sodium phosphate buffer solution at 23±2°C in the ratio established for each serotype.

Conjugation in dimethyl sulfoxide (DMSO)

For serotypes 6A, 6B, 7F, PA, 12F, 19A, 19F, and 23F, the lyophilized sample was thawed, brought to room temperature, and dissolved in DMSO.

Stage 2. Conjugation reaction

Conjugation in aqueous media

For serotypes 3-TT, 4, 5, 5-TT, 8, 9N, 9V, 10A, 14, 15B, 18C, 22F and 33F, the conjugation reaction was initiated by adding a solution of sodium cyanoborohydride (100 mg/ml) to 1.0 to 1.4 mol sodium cyanoborohydride per mole of saccharide. However, for serotypes 1, 1-TT and 3, the reaction was initiated by adding a solution of sodium cyanoborohydride to 0.5 mol of sodium cyanoborohydride per mole of saccharide.

The reaction mixture was incubated at 23 to 37°C for 44 to 106 hours. Temperature and reaction duration were adjusted for serotype. Then the temperature was reduced 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 mol. eq. sodium borohydride per mole of saccharide. The mixture was incubated at 23±2°C for 3 to 6 hours. This procedure reduced the presence of any unreacted aldehydes on the saccharides. Then

- 18 044822 the mixture was diluted with 0.9% sodium chloride and the diluted conjugation mixture was filtered using a 0.8 or 0.45 µm pre-filter.

Conjugation in DMSO medium

For capsular polysaccharides of serotypes 6A, 6B, 7F, 11A, 12F, 19A, 19F and 23F, the conjugation reaction was initiated by adding a solution of sodium cyanoborohydride (100 mg/ml) in a ratio of 0.8 to 1.2 mol. eq. 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 for 12 to 26 h at 23 ± 2°C. 100 mg/mL sodium borohydride solution (typically 1.8 to 2.2 mol equivalents of sodium borohydride per mole of activated saccharide) and WFI (target concentration 5% v/v) were added to the reaction mixture and the mixture was incubated for from 3 to 6 hours at 23±2°C. This procedure reduced the presence of any unreacted aldehydes on the saccharides. The reaction mixture was then diluted with 0.9% sodium chloride and the diluted conjugation mixture was filtered using a 0.8 or 0.45 µm pre-filter.

Stage 3. Ultrafiltration

The diluted conjugation mixture was concentrated and diafiltered on a 100 kDa MWCO ultrafiltration filter or a 300 kDa MWCO ultrafiltration filter with a minimum of 15 volumes of 0.9% sodium chloride or buffer. In addition, the composition and pH of the buffer used in this process varied among each serotype.

Stage 4. Sterile filtration

The retentate after ultrafiltration was sterile filtered (0.2 μm) and in-process controls were performed on the filtered conjugates (appearance, free protein, free saccharide, molecular size distribution, sterility, saccharide content, protein content, pH, endotoxin, residual cyanide, residual DMSO, saccharide identity, TT identity and CRM identity ₁₉₇ ). The finished concentrate was cooled and stored at 2 to 8°C.

Example 3. Preparation of a polyvalent anti-pneumococcal conjugate vaccine preparation

Desired volumes of finished bulk concentrates obtained from Example 2 were calculated based on batch size and bulk saccharide concentrations. After adding 0.85% sodium chloride (physiological saline), polysorbate 80, and succinate buffer to a pre-labeled formulation container, bulk concentrates were added to the container. The preparation was then thoroughly mixed and sterile filtered through a 0.2 µm membrane. The prepared bulk preparation was carefully stirred before and after the addition of the aluminum phosphate intermediate. If necessary, the pH was checked and adjusted. The prepared bulk product was stored at 2 to 8°C. The following non-limiting preparations of polyvalent anti-pneumococcal conjugate vaccines were prepared and named them PCV21(1/5)-TT and PCV21(3/5)-TT: PCV21(1/5)-TT included polysaccharide conjugates obtained by conjugating each polysaccharide of serotypes 1 and 5 with TT and each polysaccharide of serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F with CRM ₁₉₇ ; and PCV21(3/5)-TT included polysaccharide conjugates obtained by conjugating each polysaccharide of serotypes 3 and 5 with TT and each polysaccharide of serotypes 1, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F with CRM _W.

The PCV21(1/5)-TT formulation in a total dose of 0.5 ml included 2.2 μg of each polysaccharide, except for serotype 6B at 4.4 μg; 2 to 25 μg TT (for serotypes 1 and 5) and 40 to 75 μg CRM ₁₉₇ ; 0.125 mg elemental aluminum adjuvant (0.5 mg aluminum phosphate); 4.25 mg sodium chloride; about 295 μg of succinate buffer solution and about 100 μg of polysorbate 80 in a total dose of 0.5 ml. In some experiments, the amount of serotype 3 polysaccharide in the PCV21(1/5)-TT composition was increased to 4.4 μg or 8.8 μg. In some experiments, the amount of polysaccharide of each of serotypes 3, 4, 6B, 9V, 19A and 19F in the PCV21(1/5)-TT composition was increased to 4.4 μg.

The PCV21(3/5)-TT composition in a total dose of 0.5 ml included from 2 to 25 μg TT (for serotypes 3 and 5) and from 40 to 75 μg CRM ₁₉₇ , respectively, while the remaining components and their contents are identical to those for PCV21(1/5)-TT. In some compositions, the amount of elemental aluminum in a dose of 0.5 ml was increased to 0.250 mg.

Example 4: Immunogenicity of polyvalent pneumococcal conjugate vaccine

The mixed-carrier multivalent pneumococcal vaccines, PCV21(1/5)-TT and PCV21(3/5)-TT, prepared in Example 3 were tested for their ability to induce an immunogenic response in rabbits. Immunogenicity was assessed using antigen-specific solid-phase ELISA for IgG concentration in blood serum and opsonophagocytic assay (OPA) for antibody functionality.

New Zealand White rabbits at week 0 and week 2 were immunized intramuscularly with a dose 5% higher than the planned human clinical dose of each polysaccharide (2.31 μg of each polysaccharide, except 6B at 4.62 μg) in the formulation, or the human dose (2.2 µg of each polysaccharide, except 6B at 4.4 µg). Serum was collected every 2 weeks after immunization. Both

- 19 044822 concentrations showed the same results.

4-1. PCV21(3/5)-TT

Measurement of serotype-specific IgG concentrations

Capsular polysaccharides (PnP) for each serotype were applied to a 96-well plate at 0.5 μg/well to 1 μg/well. An equivalent amount of serum was sampled from each subject and pooled by group. The pooled serum was serially diluted 2.5-fold with antibody dilution buffer containing Tween 20 and pneumococcal cell wall polysaccharide (CWPS) obtained from the Danish National Serum Institute (5 μg/ml) and then reacted at room temperature for 30 min. . The plate was washed 5 times with wash buffer, and then 50 μl of pre-adsorbed and diluted serum was added to the coated wells of the plate, followed by incubation at room temperature for 2 to 18 hours. The plate wells were washed in the same manner and then conjugates were added to each well goat anti-rabbit IgG antibodies with alkaline phosphatase, and then incubated at room temperature for 2 hours. The plates were washed as described above and buffer with 1 mg/ml pnitrophenylamine as substrate was added to each well and then the reaction was carried out at room temperature for 2 h. The reaction was quenched by adding 50 μl of 3 M NaOH and absorbance was measured at 405 nm and 690 nm. As a comparative example, a commercially available 13valent vaccine (PREVNAR13) can be subjected to the same procedure. The results are presented in table. 3.

Table 3. IgG concentration (U/ml) for 21 serotypes 2 weeks after secondary immunization

Serotype PREVNAR13 PCV21(3/5)-TT 0.125 mg A1 PCV21(3/5)-TT 0.250 mg A1 1 16770.4 15320.6 38499.1 3 6603.4 30132.8 32029.7 4 27969.9 51034.4 71638.3 5 5758.6 15627.5 11044.7 6A 9493.7 14205.8 30453.5 6V 8690.6 15023.6 11633.1 7F 60819.7 53696.7 48511.1 8 594.8 58391.9 62852.2 9N 5186.2 208401.8 259323.2 9V 30043.9 38143.9 48677.2 10A 169.8 36437.6 55124.5 ON 184.5 36771.1 45762.9 12F 130.0 22278.5 14475.3 14 21906.0 37577.9 64409.8 15V 843.5 22065.2 25248.8 18C 91500.7 88824.7 137638.5 19A 16470.7 13070.9 11387.0 19F 13956.4 40516.9 84553.8 22F 139.7 51649.9 62072.7 23F 12089.4 5772.9 15565.5 33F 143.2 35602.4 46272.8

When capsular polysaccharides of serotypes 3 and 5 were conjugated to TT, the concentration of serotype-specific IgG increased significantly compared to the concentration obtained when they were conjugated to CRM ₁₉₇ . Rabbits immunized with PCV21(3/5)-TT also showed significant increases in IgG concentrations against an additional eight serotypes not present in PREVNAP13 (i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F). Serotype 9N in particular, when administered with 0.125 mg and 0.250 mg aluminum, gave an unexpected 40-fold and 50-fold increase, respectively, in serum concentrations of specific IgG relative to PREVNAR13. Additionally, a number of other capsular polysaccharide serotypes in PCV21(3/5)-TT showed significantly higher serum concentrations of specific IgG than PREVNAR13.

Functional Immunogenicity Testing (MOTA)

Antibody functions were assessed by testing serum in the MOPA assay. S. pneumoniae strain MOPA, stored at -70°C or below, was diluted to the appropriate final dilution factor such that the concentration of each strain was approximately 50,000 CFU/ml. An equivalent amount of serum was sampled from each subject, pooled across groups, and diluted in a 2-fold series such that 20 μl of serum remained in a U-bottom dish. After diluting the sample, 10 µl of the strain prepared for each serotype was mixed with

- 20 044822 diluted sample and the mixture was allowed to react at room temperature for 30 minutes to allow the S. pneumoniae and antibody to mix well. A mixture of pre-differentiated HL-60 cells was added and the reaction was carried out in a CO ₂ incubator (37°C) for 45 minutes. The temperature was reduced to stop phagocytosis and 10 μl of the reaction solution was placed on an agar plate that had been previously dried for 30 to 60 min and then allowed to soak on the plate for 20 min until dry. A 25 mg/ml TTC stock solution was added to the prepared layer agar and an antibody suitable for the respective strain was added to it. The mixture was stirred thoroughly and then about 25 ml of the mixture was added to the cup and allowed to harden for about 30 minutes. The completely solidified dish was incubated in a CO ₂ incubator (37°C) for 12 to 18 hours, after which the colonies were counted. The MOPA titer was expressed as the dilution factor at which 50% mortality was observed. As a comparative example, a commercially available 13-valent vaccine (PREVNAR13) can be subjected to the same procedure. The results are presented in table. 4.

Table 4. MOPA titers for 21 serotypes 2 weeks after secondary immunization

Serotype PREVNAR13 PCV21(3/5)-TT 0.125 mg A1 PCV21(3/5)-TT 0.250 mg A1 1 94 72 244 3 829 740 3515 4 2428 2698 2675 5 1169 4154 2735 6A 4925 4567 4761 6V 5693 4959 6629 7F 2731 2095 2103 8 Not tested 710 731 9N Not tested 4660 1371 9V 271 280 254 10A Not tested 952 935 ON Not tested 743 1053 12F Not tested 846 737 14 1917 2119 2170 15V Not tested 713 849 18C 5347 2620 4546 19A 5760 2464 2253 19F 2059 2071 2137 22F Not tested 6022 7783 23F 1975 1487 1566 33F Not tested 824 1924

When serotypes 3 and 5 were conjugated to TT, functional MOPA titers increased significantly compared to MOPA titers obtained when they were conjugated to CRM ₁₉₇ . Rabbits immunized with PCV21(3/5)-TT also showed significant increases in functional MOPA titers against each of the additional eight serotypes not present in PREVNAP13 (i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F). .

4-2. PCV21(1/5)-TT

Serotype-specific IgG concentrations and functional immunogenicity titers were measured in the same manner as in 4-1 and the results are shown below. Three different embodiments of PCV21(1/5)-TT were tested: one with 2.2 μg of capsular polysaccharide from serotype 3 (3CRM ₁₉₇ 2.2), one with 4.4 μg of capsular polysaccharide from serotype 3 (3-CRM ₁₉₇ 4.4) and one with 8.8 μg of capsular polysaccharide from serotype 3 (3-CRM197 8.8).

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Measurement of serotype-specific IgG concentrations

Table 5. IgG concentration (U/ml) for 21 serotypes 2 weeks after secondary immunization

Serotype PREVNAR13 PCV21(l/5)-TT 3-CRM197 2.2 PCV21(l/5)-TT 3-CRM197 4.4 PCV21(l/5)-TT 3-CRM197 8.8 1 5105.5 33530.6 96552.2 44714.9 3 6303.0 7827.2 14676.2 13239.6 4 46727.3 58893.8 54382.8 39691.0 5 6873.9 14918.4 18743.2 11499.5 6A 32561.4 11493.2 13053.0 12494.6 6V 25398.7 11856.7 10013.2 4455.6 7F 27560.5 25799.0 70917.5 56448.2 8 521.0 70060.4 86634.8 63337.9 9N 5198.2 169584.3 240858.5 187203.6 9V 62169.3 42445.1 64084.4 34876.4 10A 166.4 25682.8 44186.9 15827.4 PA 195.3 34050.4 42558.8 33264.2 12F 154.4 27629.4 38459.7 25874.3 14 17765.9 23787.4 29262.4 24116.4 15V 436.0 22060.6 21777.0 22573.1 18C 103154.7 119672.1 141889.4 75549.1 19A 19191.1 4690.5 5516.9 3407.3 19F 16349.2 31789.8 24370.0 18846.2 22F 130.0 40963.1 49475.5 36246.2 23F 15166.5 6312.7 7499.9 5431.5 33F 146.1 39803.9 38378.5 26837.2

When capsular polysaccharides of serotypes 1 and 5 were conjugated to TT, the concentration of serotype-specific IgG increased significantly compared with the concentration obtained when they were conjugated to CRM ₁₉₇ . Rabbits immunized with PCV21(1/5)-TT also showed significant increases in IgG concentrations against an additional eight serotypes not present in PREVNAP13 (i.e., 8, 9N, 10A, 11 A, 12F, 15B, 22F, and 33F). Again, serotype 9N unexpectedly showed a significant increase (approximately 32- to 46-fold) compared to PREVNAR13. Additionally, a number of other capsular polysaccharide serotypes in PCV21(1/5)-TT showed significantly higher serum specific IgG concentrations than PREVNAR13. It was also observed that doubling the amount of serotype 3 antigen from 2.2 μg to 4.4 μg unexpectedly increased the serum concentration of specific IgG for serotypes 1, 3, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 18C, 19A, 22F and 23F, with more than a 2-fold increase for serotypes 1 and 7F. Functional Immunogenicity Testing (MOTA)

Table 6. MOPA titers for 21 serotypes 2 weeks after secondary immunization

Serotype PREVNAR13 PCV21(l/5)-TT 3-CRM197 2.2 PCV21(l/5)-TT 3-CRM197 4.4 PCV21(l/5)-TT 3-CRM197 8.8 1 99 198 916 241 3 479 565 1072 929 4 2560 3201 2571 2012 5 1046 3717 5430 2025 6A 5624 2708 5249 2415

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6V 5451 3903 4143 2452 7F 2355 2521 2576 2125 8 Not tested 525 719 630 9N 54 790 1680 885 9V 282 251 307 183 10A Not tested 684 1065 760 ON Not tested 887 1751 747 12F Not tested 856 1016 824 14 1052 1221 1929 1513 15V 59 1042 823 732 18C 6257 4440 4663 2821 19A 2962 828 1362 686 19F 968 1971 1835 1453 22F Not tested 3576 5599 7193 23F 1854 769 1358 1050 33F Not tested 932 1982 808

When serotypes 1 and 5 were conjugated to TT, functional MOPA titers increased significantly compared to MOPA titers obtained when they were conjugated to CRM ₁₉₇ . Rabbits immunized with PCV21(1/5)-TT also showed significant increases in functional MOPA titers against each of the additional eight serotypes not present in PREVNAP13 (i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F). . Additionally, several other serotypes showed significantly higher levels of functional MOPA titers than PREVNAR13. It was also observed that doubling the amount of serotype 3 antigen from 2.2 μg to 4.4 μg unexpectedly increased the serum concentration of specific IgG for every serotype except 4, 15B, and 19F, with a greater than 4.5-fold increase for serotype 1. 4-3. PCV21(1/5)-TT

Experiments 4-2 were repeated with an embodiment of PCV21(1/5)-TT containing 2.2 μg of capsular polysaccharide from serotype 3 (3-CRM ₁₉₇ 2.2), an embodiment of PCV21(1/5)-TT containing 4 .4 μg capsular polysaccharide from serotype 3 (3-CRM ₁₉₇ 4.4), and an embodiment PCV21(1/5)-TT containing 4.4 μg capsular polysaccharide from serotypes 3, 4, 6B, 9V, 19A and 19F (multi-CRM ₁₉₇ 4.4). Serotype-specific IgG concentrations and functional immunogenicity titers were measured in the same manner as in 4-1 and the results are shown below.

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Measurement of serotype-specific IgG concentrations

Table 7. IgG concentration (U/ml) for 21 serotypes 2 weeks after secondary immunization

Serotype PREVNAR13 PCV21(l/5)-TT 3-CRM197 2.2 PCV21(l/5)-TT 3CRM197 4.4 PCV21(l/5)-TT Mulyp-CRM 197 4.4 1 14208.3 34503.1 49226.1 40684.4 3 6575.6 6634.1 13769.4 10841.3 4 16600.3 33352.0 43605.9 34387.8 5 5079.3 10847.5 20866.2 13405.1 6A 8965.5 2607.1 13781.5 9194.9 6V 5105.2 1233.5 9225.5 2428.9 7F 59993.0 27961.7 57039.5 33913.6 8 274.7 56735.3 72822.9 65528.5 9N 3824.7 140424.7 181325.9 142736.9 9V 39503.5 29139.3 61759.9 48711.5 10A 130.0 8017.0 14061.8 15832.8 ON 163.9 33051.7 51222.1 39311.8 12F 130.0 18160.9 22634.9 14877.6 14 12312.4 12868.7 15953.4 8538.5 15V 280.6 14339.7 22347.7 22692.0 18C 62963.5 56059.2 141911.5 70910.8 19A 9807.5 1185.7 4233.4 2649.2 19F 9838.7 9825.8 21175.9 10889.9 22F 130.0 20120.0 37833.7 21807.5 23F 5835.1 2084.1 4016.2 3152.3 33F 141.4 22732.6 19748.9 19179.6

As in 4-2, when capsular polysaccharides of serotypes 1 and 5 were conjugated to TT, the concentration of serotype-specific IgG increased significantly compared with the concentration obtained when they were conjugated to CRM ₁₉₇ . Rabbits immunized with PCV21(1/5)-TT also showed significant increases in IgG concentrations against an additional eight serotypes not present in PREVNAP13 (i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F), and capsular polysaccharide serotype 4 showed significantly higher serum concentrations of specific IgG than PREVNAR13. It was also observed that doubling the amount of serotype 3 antigen from 2.2 μg to 4.4 μg unexpectedly increased serum concentrations of specific IgG for all three serotypes. It was also possible to use a higher dose of multiple serotypes, as demonstrated by embodiment PCV21(1/5)-TT, in which the number of serotypes 3, 4, 6B, 9V, 19A and 19F was increased from 2.2 μg to 4.4 μg (multi-CRM ₁₉₇ 4.4).

Functional Immunogenicity Testing (MOTA)

Table 8. MOPA titers for 21 serotypes 2 weeks after secondary immunization

Serotype PREVNAR13 PCV21(l/5-TT) 3-CRM197 2.2 PCV21(l/5-TT) 3-CRM197 4.4 PCV21(l/5-TT) Multi-SKM197 4.4 1 109 236 276 255 3 740 514 836 768

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4 2272 2522 2216 2376 5 3638 12393 18293 2559 6A 4949 2187 3607 2401 6V 4915 767 5196 1555 7F 2414 2014 2227 1781 8 Not tested 582 694 690 9N 84 937 1252 983 9V 295 474 419 290 10A Not tested 566 737 674 ON Not tested 801 1666 1878 12F Not tested 600 1008 1298 14 1659 1503 1200 959 15B 79 902 1459 1794 18C 2933 2594 4095 2968 19A 3910 957 1244 751 19F 1570 908 2175 1453 22F Not tested 2268 7833 3516 23F 1956 677 786 709 33F Not tested 1021 1115 736

As in 4-2, when serotypes 1 and 5 were conjugated to TT, functional immunogenicity was improved compared with the immunogenicity obtained when they were conjugated to CRM ₁₉₇ . Rabbits immunized with PCV21(1/5)-TT also showed significant increases in functional MOPA titers against each of the additional eight CRM ₁₉₇ -conjugated serotypes not present in PREVNAR13 (i.e., 8, 9N, 10A, 11A, 12F, 15B , 22F and 33F). It was also possible to use a higher dose of multiple serotypes, as demonstrated for PCV21(1/5)-TT multi-CRM ₁₉₇ 4.4, in which the number of serotypes 3, 4, 6B, 9V, 19A and 19F was increased from 2.2 mcg to 4.4 mcg.

4-4. PCV21(1/5)-TT

Experiments 4-2 were repeated with an embodiment of PCV21(1/5)-TT containing 4.4 μg of capsular polysaccharide from serotypes 3 and 4 (3,4-CRM ₁₉₇ 4,4). Serotype-specific IgG concentrations and functional immunogenicity titers were measured in the same manner as in 4-1 and the results are shown below. Measurement of serotype-specific IgG concentrations

- 25 044822

Table 9. IgG concentration (U/ml) for 21 serotypes 2 weeks after secondary immunization for 3,4-CRM ₁₉₇ 4.4

Serotype Prevnar13 PCV21 (1.5-TT) 3.4-CRMi97 4.4 1 2512.6 11687.9 3 6160.3 16537.5 4 13018.1 23834.9 5 7738.6 35815.1 6A 13701.1 6476.3 6V 2290.0 881.1 7F 22885.1 25523.5 8 170.1 19027.5 9N 971.4 35064.1 9V 13803.2 18139.3 10A 130.0 7028.4 ON 165.6 10215.7 12F 130.0 3770.0 14 3333.0 4977.4 15V 203.2 8956.5 18C 28120.5 21748.1 19A 18587.6 6785.8 19F 29732.6 34497.2 22F 130.0 21193.6 23F 3962.9 1791.5 33F 130.0 13194.5

When capsular polysaccharides of serotypes 1 and 5 were conjugated to TT, the concentration of serotype-specific IgG increased significantly compared with the concentration obtained when they were conjugated to CRM ₁₉₇ . Rabbits immunized with PCV21(1/5)-TT also showed significant increases in IgG concentrations against an additional eight serotypes not present in PREVNAP13 (i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F). It was also observed that doubling the amount of serotype 3 and serotype 4 antigen from 2.2 μg to 4.4 μg showed significantly higher serum specific IgG concentrations than PREVNAR13. It was also possible to use a higher dose of multiple serotypes, as demonstrated by embodiment PCV21(1/5)TT, in which the number of serotypes 3 and 4 was increased from 2.2 μg to 4.4 μg (3,4-CRM ₁₉₇ 4, 4).

Functional Immunogenicity Testing (MOTA)

Table 10. MOPA titers for 21 serotypes 2 weeks after secondary immunization

Serotype Prevnar13 PCV21 (1.5-TT) 3.4-CRMi97 4.4 1 54 455 3 393 968

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4 2072 3331 5 306 1621 6A 2355 1315 6V 1614 1410 7F 952 981 8 5 986 9N 52 2072 9V 324 479 10A 2 427 11A 4 1103 12F 2 266 14 539 969 15V 5 255 18C 1996 1392 19A 1870 774 19F 1516 1352 22F 2 1796 23F 928 496 33F 2 292

When serotypes 1 and 5 were conjugated to TT, functional immunogenicity was improved compared with the immunogenicity obtained when they were conjugated to CRM ₁₉₇ . Rabbits immunized with PCV21(1/5)-TT also showed significant increases in functional MOPA titers against each of the additional eight CRM197-conjugated serotypes not present in PREVNAR13 (i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F and 33F). It was also possible to use a higher dose of multiple serotypes, as demonstrated by PCV21(1/5)-TT 3,4-CRM ₁₉₇ 4,4, in which the number of serotypes 3 and 4 was increased from 2.2 μg to 4.4 μg.

Example 5 Additional Information on Preparation of Serotype 9N Streptococcus pneumoniae Saccharide-Protein Conjugate Preparation of a Cell Bank

Streptococcus pneumoniae serotype 9N (ATCC 6309) was obtained from the American Type Culture Collection (ATCC). To propagate the strain and remove components of animal origin, the seed culture was cultivated for several generations. The vial with the seed culture was stored in the refrigerator (< -70°C) along with synthetic glycerol as a cryoprotectant. To obtain a cell bank, the cell culture was propagated in soybean medium. Before freezing, the cells were concentrated by centrifugation and, after removal of the depleted medium, the pelleted cells were resuspended in fresh medium containing a cryoprotectant (such as synthetic glycerol).

Fermentation

The culture from the cell bank was inoculated into a seed bottle containing soybean medium. Until satisfactory growth conditions were achieved, the culture was incubated at a constant temperature without stirring. Using a seed bottle, the culture was inoculated into a seed fermenter containing soybean media with controlled temperature, pH, and agitation speed. Fermentation was stopped when growth stopped or the working capacity of the fermenter was reached. After fermentation was completed by adding a deactivator, cell debris was removed using a combination of continuous flow centrifugation and filtration.

Cleaning

The purification process of pneumococcal polysaccharides consisted of multi-layer filtration, multiple concentration/diafiltration and filtration/elution.

Activation

The final polysaccharide concentration was adjusted to about 2.0 g/L by sequentially adding the calculated amount of WFI. If necessary, the pH of the reaction was adjusted to approximately 6.0. After adjusting the pH, the reaction temperature was set to 21-25°C. To initiate oxidation, about 0.024-0.189 mg of sodium periodate was added per 1 mg of sugar. The oxidation reaction was carried out for 16-20 hours at 21-25°C.

The activated polysaccharide was concentrated and filtered using a 100 kDa MWCO ultrafiltration membrane. Diafiltration was carried out with WFI 10 times the volume of diafiltration. The purified activated polysaccharide was then stored at 2-8°C.

The purified activated saccharide was characterized by (i) saccharide concentration as determined by colorimetric analysis, (ii) aldehyde concentration as determined by colorimetric analysis, (iii) degree of oxidation, and (iv) molecular weight as measured by SEC-MALLS.

EX-MALLS is used to determine the molecular weight of polysaccharide and polysaccharide-protein conjugates. SEC is used to separate polysaccharides based on hydrodynamic volume. To determine the molecular weight, a refractive index detector (RI) and a multi-angle laser light scattering detector (MALLS) are used. When light reacts with matter, it scatters. The amount of scattered light is related to the concentration, the square of dn/dc (specific increase in refractive index) and the molar mass of the substance.

The molecular weight is calculated from the scattered light signal from the MALLS detector and the concentration signal from the RI detector.

The degree of oxidation (DO) of an activated polysaccharide is determined as the moles of sugar repeat units divided by the moles of aldehyde. The moles of sugar repeat units are determined by various colorimetric techniques, for example using the anthrone assay.

And, aldehyde moles are determined by Park-Johnson colorimetric assay.

Using the methods described above, it was determined that the activated capsular polysaccharide of Streptococcus pneumoniae serotype 9N, obtained by the method described above, has an oxidation number of 2-19, usually 5-10, and a molecular weight of about 200-700 kDa.

Conjugation

The activated polysaccharide was mixed with the CRM ₁₉₇ carrier protein in a ratio of 0.5-2 g of CRM ₁₉₇ per 1 g of activated polysaccharide. The combined mixture was then lyophilized. The lyophilized mixture of activated polysaccharide and CRM197 was stored at -20°C.

The lyophilized mixture of activated polysaccharide and CRM197 was dissolved in 0.1 M sodium phosphate solution and then mixed sufficiently. The final concentration of polysaccharides in the reaction solution was about 10-20 g/l. After initiation of conjugation by adding 1.0-1.2 mol. to the reaction mixture. eq. sodium cyanoborohydride (NaBH3CN), the reaction was carried out at 3539°C for 44-52 hours. The conjugation reaction was completed by adding a 0.9% sodium chloride solution of the same volume as the conjugation reaction solution, and then adding 1.8-2.2 mol . eq. sodium borohydride (NaBH ₄ ) to block unreacted aldehyde. The blocking reaction was carried out at 21-25°C for 3-6 hours.

The conjugate solution was diluted with 0.9% sodium chloride solution for concentration and diafiltration using a 100 kDa MWCO membrane. The diluted solution of conjugates was filtered through a 0.8-0.45 μm filter and purified by concentration and diafiltration. Diafiltration was carried out using a 100 kDa MWCO membrane using a 0.9% sodium chloride solution with a volume of 15-40 times the diafiltration volume. After completion of diafiltration, the remaining solution was filtered through a 0.2 μm filter. The conjugate solution was diluted to a concentration of less than about 0.55 mg/ml, sterile filtered and then stored at 2-8°C.

The characteristics of the purified serotype 9N conjugate were determined, in particular, by (i) protein concentration determined by colorimetric analysis (Lowry), (ii) aldehyde concentration determined by colorimetric analysis, (iii) saccharide-protein ratio, (iv) molecular size distribution by method size exclusion chromatography (CL-4B) and (v) molecular weight measured by SEC-MALLS. Changes in the characteristics of the serotype 9N conjugate were observed when the degree of oxidation (DO) was varied. The result is shown in table. eleven.

Table 11

Conjugate number 1 2 3 4 5 6 Molecular weight of activated polysaccharide, kDa 582 619 459 563 490 427 DO 18.2 9.4 7.4 6.7 4.3 2.3 Initial ratio (P:S) 0.8:1 Concentration of polysaccharide in the conjugation reaction solution, g/l 20.0 % yield of conjugate 53 43 39 32 33 39 Saccharide-protein ratio 2.1 1.5 1.3 1.1 1.0 0.78 % free saccharide 44 28 22 20 21 31 % molecular weight distribution 52 49 50 55 44 31 Molecular mass of the conjugate, kDa 860 1 110 1,912 1 168 1 189 1 160

A change in the characteristics of the serotype 9N conjugate was observed by varying the mixing ratio of the activated polysaccharide and CRM197 during conjugation. The result is shown in table. 12.

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Table 12

Conjugate number 7 8 9 10 eleven Molecular weight of activated polysaccharide, kDa 287 DO 5.6 Initial ratio (P:S) 2:1 1.5:1 1:1 0.67:1 0.5:1 Concentration of polysaccharide in the conjugation reaction solution, g/l 20.0 % yield of conjugate 25 50 43 41 66 Saccharide-protein ratio 0.71 0.85 1.0 1.2 1.8 % free saccharide 5 6 15 27 62 % molecular weight distribution 52 58 50 40 22 Molecular mass of the conjugate, kDa 3 720 3,713 1 327 1,016 545

A change in the characteristics of the serotype 9N conjugate was observed when the concentration of polysaccharides in the conjugation reaction solution was varied. The result is shown in Table 13.

Table 13

Conjugate number 12 13 14 15 16 Molecular weight of activated polysaccharide, kDa 560 DO 6.1 Initial ratio (P:S) 0.8:1 Concentration of polysaccharide in the conjugation reaction solution, g/l 10.0 12.5 15.0 17.5 20.0 % yield of conjugate 20 31 28 40 42 Saccharide-protein ratio 1.0 1.0 0.93 0.99 0.97 % free saccharide 32 thirty 22 21 18 % molecular weight distribution 17 27 40 47 54 Molecular mass of the conjugate, kDa 560 546 845 932 1438

Example 6 Immunogenicity Assay

A monovalent conjugated composition was prepared containing a saccharide-protein conjugate of Streptococcus pneumoniae serotype 9N conjugated to CRM ₁₉₇ .

Immunogenicity of monovalent immunogenic compositions table. 11-13 were analyzed by solid-phase ELISA. The serum concentration of serotype-specific IgG was determined.

Five female New Zealand White rabbits weighing 2.5-3.5 kg were immunized with the proposed human clinical dose (2.2 μg conjugate; + 0.25 mg/ml aluminum as AIPO4) at week 0 via the intramuscular route. Rabbits were immunized again at week 2 with the same dose of conjugate vaccine and blood samples were collected at week 4. Serotype-specific ELISA was performed on week 0 and week 4 serum samples.

The result of the analysis is shown in table. 14. In rabbits immunized with the monovalent conjugate composition (conjugate number 8), there was a significant increase in total IgG titer for serotype 9N. Rabbits immunized with other conjugates also showed a significant increase in total IgG titer.

In table Figure 14 shows the result of measuring the concentration of IgG after immunization of rabbits with conjugate number 8 from table. 12.

Table 14

IgG concentration (U/ml) Serotype Pre-immunization Subsequent immunization 9N 130.0 656,345.3

While one or more exemplary embodiments have been described herein, one of ordinary skill in the art will appreciate that various changes in form and detail may be made herein without departing from the spirit and scope of the invention as defined by the following claims.

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Claims (19)

Literature The following references are cited in this application and provide general information regarding the technical field and also provide descriptions of the assays and other details discussed in this application. The following references are incorporated herein by reference in their entirety. 1. A multivalent anti-pneumococcal mixed-carrier conjugate composition comprising 21 different pneumococcal capsular polysaccharide-protein conjugates, each pneumococcal capsular polysaccharide-protein conjugate containing a protein carrier conjugated to a capsular polysaccharide from different serotypes of Streptococcus pneumoniae, wherein the serotypes Streptococcus pneumoniae selected from 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein the protein carrier is CRM ₁₉₇ or tetanus toxoid, wherein two of the capsular polysaccharides are conjugated to tetanus toxoid and the remaining capsular polysaccharides are conjugated to CRM ₁₉₇ , wherein the two capsular polysaccharides that are conjugated to tetanus toxoid are selected from the group consisting of serotypes 1, 3 and 5. [1] Prymula et al., The Lancet, 367:740-48 (2006). 2. Polyvalent anti-pneumococcal conjugated composition with a mixed carrier according to claim 1, in which capsular polysaccharides from serotypes 1 and 5 are conjugated with tetanus toxoid, and capsular polysaccharides from serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A , 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F are conjugated to CRM ₁₉₇ . [2] Vesikari et al., PIDJ, 28(4):S66-76 (2009). 3. Polyvalent anti-pneumococcal conjugated composition with a mixed carrier according to claim 1, in which capsular polysaccharides from serotypes 1 and 3 are conjugated with tetanus toxoid, and capsular polysaccharides from serotypes 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A , 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F are conjugated to CRM ₁₉₇ . [3] Dagan et al., Infection & Immunity, 5383-91 (2004). 4. Polyvalent anti-pneumococcal conjugated composition with a mixed carrier according to claim 1, in which capsular polysaccharides from serotypes 3 and 5 are conjugated with tetanus toxoid, and capsular polysaccharides from serotypes 1, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A , 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F are conjugated to CRM ₁₉₇ . [4] Juergens et al., Clinical and Vaccine Immunology, 21(9): 1277-1281 (2014). 5. Multivalent anti-pneumococcal conjugate composition with a mixed carrier according to any of the preceding paragraphs, additionally containing an adjuvant. [5] Andrews et al., The Lancet, 14:839-846 (2014). 6. The multivalent anti-pneumococcal mixed-carrier conjugate composition according to claim 5, wherein the adjuvant is an aluminum-based adjuvant. [6] Nurkka et al., Vaccine, 20:194-201 (2001). 7. The multivalent anti-pneumococcal mixed-carrier conjugate composition according to claim 6, wherein the adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide. [7] Levin and Stone, J. Immunol., 67:235-242 (1951). 8. The multivalent anti-pneumococcal mixed-carrier conjugate composition according to claim 7, wherein the adjuvant is aluminum phosphate. [8] W.H.O. Manual for the Production and Control of Vaccines: Tetanus Toxoid, 1977 (BLG/UNDP/77.2 Rev.I.) 9. The multivalent anti-pneumococcal mixed-carrier conjugate composition according to any one of the preceding claims, wherein the capsular polysaccharide from serotype 9N is conjugated to CRM ₁₉₇ in a state in which the capsular polysaccharide from serotype 9N is activated to obtain an oxidation state of 2-19 or 5-10 and molecular masses 200-700 kDa; the conjugate formed between the capsular polysaccharide from serotype 9N and CRM ₁₉₇ has a molecular weight of 500-4000 kDa; the ratio (w/w) of capsular polysaccharide from serotype 9N and CRM ₁₉₇ in the conjugate formed between capsular polysaccharide from serotype 9N and CRM ₁₉₇ is 0.5-2.5; and/or [9] Didierlaurent et al., J. Immunol., 183:6186-6197 (2009). CLAIM 10. The use of a multivalent anti-pneumococcal conjugate composition with a mixed carrier according to any one of the preceding claims for the prevention of Streptococcus pneumoniae infection or disease in a subject. 11. Use according to claim 10, wherein the subject is a human. 12. A vaccine containing a multivalent anti-pneumococcal conjugate composition with a mixed carrier according to any one of claims 1 to 9 and a pharmaceutically acceptable excipient. 13. A method of preventing a Streptococcus pneumoniae infection or disease in a subject, comprising administering to the subject a prophylactically effective amount of a multivalent anti-pneumococcal conjugate mixed-carrier composition according to any one of claims 1 to 9 or a vaccine according to claim 12. 14. The method of claim 13, wherein the subject is a person at least 50 years of age and the disease is pneumonia or invasive pneumococcal disease (IPD). 15. The method of claim 13, wherein the subject is a human at least 6 weeks old and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM). 15-60% of the conjugate formed between the capsular polysaccharide from serotype 9N and CRM ₁₉₇ has a Kd value of 0.3 or lower on the CL-4B column. - 30 044822 16. The method of claim 15, wherein the subject is between 6 weeks and 5 years, 2 and 15 months, or 6 and 17 years. 17. The method of claim 13, wherein the subject is a human. 18. The method according to any one of claims 13 to 17, wherein the multivalent anti-pneumococcal mixed-carrier conjugate composition or vaccine is administered by intramuscular injection. 19. The method according to any one of claims 13 to 18, wherein the multivalent anti-pneumococcal mixed-carrier conjugate composition or vaccine is administered as part of a series of immunizations. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2