Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof

Abstract

The present invention relates to novel immunogenic compositions comprising conjugated Streptococcus pneumoniae capsular saccharide antigens (glycoconjugates) and uses thereof. The immunogenic compositions of the invention typically comprise at least one glycoconjugate derived from a Streptococcus pneumoniae serotype not found in Prevnar, Synflorix, and / or Prevnar 13. The present invention also relates to the vaccination of human subjects, particularly infants and the elderly, against pneumococcal infection using said novel immunogenic compositions.

Description

[Technical Field]

[0001] The present invention relates to novel immunogenic compositions comprising conjugated capsular saccharide antigens (glycoconjugates) and uses thereof. The immunogenic compositions of the invention typically comprise glycoconjugates, where the saccharide is derived from a serotype of Streptococcus pneumoniae. The invention also relates to the vaccination of human subjects, particularly infants and the elderly, against pneumoccocal infection using the novel immunogenic compositions. [Background technology]

[0002] Infections caused by Streptococcus pneumoniae are a major cause of morbidity and mortality worldwide. Pneumonia, febrile bacteremia, and meningitis are the most common symptoms of invasive pneumococcal disease, but bacteria spread within the respiratory tract can lead to middle ear infections, sinusitis, or recurrent bronchitis. Compared to invasive disease, non-invasive symptoms are usually less severe but are significantly more common.

[0003] In Europe and the United States, pneumococcal pneumonia is the most common community-acquired bacterial pneumonia, estimated to affect approximately 100 per 100,000 adults annually. The corresponding numbers for febrile bacteremia and meningitis are 15-19 per 100,000 and 1-2 per 100,000, respectively. The risk of one or more of these conditions is significantly higher in infants and the elderly, as well as immunocompromised individuals of any age. Even in economically developed areas, invasive pneumococcal disease carries a high mortality rate; for adults with pneumococcal pneumonia, mortality averages 10%-20%, whereas in high-risk groups, it can exceed 50%. Pneumonia is by far the most common cause of pneumococcal deaths worldwide.

[0004] Streptococcus pneumoniae (the pneumococcus), the causative agent of pneumococcal disease, is a Gram-positive, encapsulated cocci surrounded by a polysaccharide capsule. Differences in the composition of this capsule allow serological differentiation among approximately 91 capsular types, some of which are frequently associated with pneumococcal disease, while others are rare. Invasive pneumococcal infections include pneumonia, meningitis, and febrile bacteremia; common noninvasive symptoms include otitis media, sinusitis, and bronchitis.

[0005] Pneumococcal conjugate vaccine (PCV) is a pneumococcal vaccine used to protect against disease caused by Streptococcus pneumoniae (pneumococcus). There are currently five PCV vaccines available on the global market: Prevnar® (called Prevenar in some countries) (7-valent vaccine), SYNFLORIX® (10-valent vaccine), Prevnar 13® (13-valent vaccine), Vaxneuvance™ (15-valent vaccine), and Prevnar 20™ (20-valent vaccine).

[0006] The recent emergence of widespread microbial resistance to essential antibiotics and the increasing number of immunocompromised people highlight the need for a pneumococcal vaccine with even broader protection.

[0007] There is a need to address the remaining unmet medical need for pneumococcal disease coverage, particularly due to serotypes not found in Prevnar 13® and the potential for serotype replacement over time. The specific disease-causing serotypes beyond the 13 in Prevnar 13® vary by region, population, and may change over time due to the acquisition of antibiotic resistance, the introduction of pneumococcal vaccines, and persistent trends of unknown origin. There is a need for immunogenic compositions that can be used to induce immune responses against additional pneumococcal serotypes in humans, particularly in children under the age of 2 years. Summary of the Invention [Problem to be solved by the invention]

[0008] The objective of the novel immunogenic compositions of the present invention is to provide adequate protection against additional Streptococcus pneumoniae serotypes not found in Prevnar 13®. In one aspect, the objective of the immunogenic compositions of the present invention is to provide adequate protection against additional Streptococcus pneumoniae serotypes not found in REVNAR® (7-valent vaccine), SYNFLORIX®, and / or PREVNAR 13®, while maintaining immune responses to serotypes currently covered by the vaccines. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows the repeating polysaccharide structure of Streptococcus pneumoniae serotype 23A (Pn-23A) capsular polysaccharide. [Figure 2] Figure 2 shows the 1D 1H proton spectra of Pn-23A polysaccharide sized using sonication (mechanical) and acid hydrolysis for 2 hours (2 Hrs), 4 hours (4 Hrs), and 6 hours (6 Hrs). Left panel: anomeric region; center panel: increased threshold for the anomeric region; right panel: methyl region of the 1H spectrum of Pn-23A polysaccharide. Anomeric and methyl signals are annotated. [Figure 3] FIG. 3 shows the normalized intensity changes of selected resonances from each sugar in the repeating unit of the Pn-23A polysaccharide. [Figure 4] Figure 4. Right panel: 1D 31P spectrum of Pn-23A polysaccharide sized using sonication (mechanical) and acid hydrolysis for 2, 4, and 6 hours. Left panel: 2D 1H-31P HMBC spectrum of hydrolyzed (6 hours) Pn-23A polysaccharide. Phosphorus-carbon correlations in the different populations are annotated using dotted lines. [Figure 5]FIG. 5 shows the structural changes observed upon hydrolysis of Pn-23A polysaccharide. [Figure 6] Figure 6 shows the 1D 1H proton spectra of Pn-23A polysaccharide sized using sonication (Sonicated Sized), homogenization (Homogenized Sized), and hydrolysis performed for 2 hours (Hydrolyzed (2 hours)). Left panel: anomeric region; center panel: increased threshold for the anomeric region; right panel: methyl region of the 1H spectrum of Pn-23A polysaccharide. Anomeric and methyl signals are annotated. [Figure 7] FIG. 7 shows the repeating polysaccharide structure of Streptococcus pneumoniae serotype 24F (Pn-24F) capsular polysaccharide. [Figure 8] Figure 8 shows the 1D 1H proton spectrum of the Pn-24F polysaccharide after hydrolysis. The anomeric and methyl signals are annotated. The table shows excellent agreement between the normalized and predicted peak areas of the anomeric and methyl protons, except for residue E1, which suggests the loss of a Ribf sugar. [Figure 9] Figure 9 shows the 1D 1H proton spectrum of the Pn-24F polysaccharide after hydrolysis. The anomeric and methyl signals are annotated. The table shows excellent agreement between the normalized and predicted peak areas of the anomeric and methyl protons (apart from the Ribf sugar (E1)). [Figure 10-1] Figure 10 shows the 1D 1H proton spectra of Pn-24F polysaccharide that was mechanically sized (bottom spectrum) or hydrolyzed under two different conditions (top and middle spectra). The anomeric and methyl signals are annotated in the reference spectrum. The table shows the 1D 1H NMR signals of the anomeric protons. [Figure 10-2]Figure 10 shows the 1D 1H proton spectra of Pn-24F polysaccharide that was mechanically sized (bottom spectrum) or hydrolyzed under two different conditions (top and middle spectra). The anomeric and methyl signals are annotated in the reference spectrum. The table shows the 1D 1H NMR signals of the anomeric protons. [Figure 11] FIG. 11 shows the structural changes observed in the Pn-24F polysaccharide after hydrolysis. DETAILED DESCRIPTION OF THE INVENTION

[0010] 1. Glycoconjugates of the Present Invention The present invention is directed, in part, to conjugated capsular saccharide antigens (also termed glycoconjugates). For the purposes of the present invention, the term "glycoconjugate" refers to a capsular saccharide that is covalently or non-covalently conjugated to a carrier protein. In one aspect, the capsular saccharide is conjugated to the carrier protein non-covalently (e.g., via the resavidin / biotin system, see, e.g., WO2012155007, WO2020056202). Preferably, the capsular saccharide is conjugated by a covalent bond. In one aspect, the capsular saccharide is directly conjugated to the carrier protein. In a second aspect, the capsular saccharide is conjugated to the carrier protein via a spacer / linker.

[0011] The term "saccharide" may refer throughout this specification to a polysaccharide or an oligosaccharide, and includes both. In frequent embodiments, the saccharide is a polysaccharide, in particular a Streptococcus pneumoniae capsular polysaccharide. Streptococcus pneumoniae capsular saccharides can be prepared by techniques known to those skilled in the art (see, for example, the methods disclosed in US2006 / 0228380, US2006 / 0228381, US2008 / 0102498, WO2008 / 118752 and WO2020170190). Typically, capsular polysaccharides are produced by growing the respective Streptococcus pneumoniae serotype in a medium (e.g., in a soy-based medium), and the polysaccharide is then prepared from the bacterial culture. The bacterial strains of Streptococcus pneumoniae used to make the respective polysaccharides used in the glycoconjugates of the invention can be obtained from established culture collections (such as, for example, from the Streptococcal Reference Laboratory (US Centers for Disease Control and Prevention, Atlanta, GA, USA)) or from clinical specimens.

[0012] Populations of organisms (each S. pneumoniae serotype) are often scaled up from seed vials to seed bottles and passaged through one or more seed fermentors of increasing volume until a production-scale fermentation volume is reached. At the end of the growth cycle, the cells are lysed and the lysate broth is then harvested for downstream (purification) processing (see, e.g., WO2006 / 110381, WO2008 / 118752 and U.S. Patent Application Publication Nos. 2006 / 0228380, 2006 / 0228381, 2008 / 0102498 and 2008 / 0286838).

[0013] Individual polysaccharides are typically purified via centrifugation, precipitation, ultrafiltration and / or column chromatography (see, e.g., WO2006 / 110352, WO2008 / 118752 and WO2020170190).

[0014] The purified polysaccharide can be activated (e.g., chemically activated) to become reactive (e.g., directly to a carrier protein or via a linker such as an eTEC spacer) and then incorporated into a glycoconjugate of the invention, as further described herein.

[0015] The S. pneumoniae capsular polysaccharide comprises repeating oligosaccharide units that can contain up to eight sugar residues. In one aspect, the capsular saccharide of the invention may be shorter than the natural length saccharide chain of one oligosaccharide unit or repeating oligosaccharide unit, hi one aspect, the capsular saccharide of the invention is one repeating oligosaccharide unit of the relevant serotype.

[0016] In one aspect, the capsular saccharide of the invention may be an oligosaccharide, which has a small number of repeating units (usually 5-15 repeating units) and is usually derived synthetically or by hydrolysis of a polysaccharide.

[0017] In one aspect, the capsular saccharides of the invention are polysaccharides. High molecular weight capsular polysaccharides are capable of inducing a specific antibody immune response due to epitopes present on the antigen surface. Preferably, isolation and purification of high molecular weight capsular polysaccharides is contemplated for use in the conjugates, compositions and methods of the invention. 1.1 Streptococcus pneumoniae serotype 15A glycoconjugates of the present invention In one aspect, the present invention relates to Streptococcus pneumoniae serotype 15A glycoconjugates.

[0018] The structure of the Streptococcus pneumoniae serotype 15A polysaccharide is known in the art (see, e.g., Geno K et al. (2015) Clin Microbiol Rev 28:3, 871-899).

[0019] In one aspect the encapsulated S. pneumoniae serotype 15A saccharide used in the invention is a synthetic carbohydrate. However, in a preferred embodiment, the source of bacterial polysaccharides according to the present invention may be Streptococcus pneumoniae serotype 15A bacterial cells. Bacterial strains that can be used as a source of Streptococcus pneumoniae serotype 15A polysaccharides can be obtained from established culture collections (such as, for example, from the Streptococcus Reference Laboratory (US Centers for Disease Control and Prevention, Atlanta, GA, USA)) or from clinical specimens.

[0020] Serotype 15A saccharides can be obtained directly from bacteria using isolation procedures known to those skilled in the art. They can also be purchased commercially (e.g., from the American Type Culture Collection (ATCC), Manassas, Virginia, USA) (e.g., reference number ATCC(537-X)).

[0021] When serotype 15A saccharides are obtained directly from bacteria, the bacterial cells can be grown in a medium, preferably a soy-based medium. After fermentation of the bacterial cells that produce Streptococcus pneumoniae serotype 15A capsular polysaccharide, the bacterial cells can be lysed to produce a cell lysate. The serotype 15A polysaccharide can then be isolated from the cell lysate using purification techniques known in the art, including centrifugation, depth filtration, precipitation, ultrafiltration, treatment with activated carbon, diafiltration, and / or column chromatography (see, e.g., US2006 / 0228380, US2006 / 0228381, WO2008 / 118752, and WO2020170190). The purified serotype 15A capsular polysaccharide can then be used to prepare glycoconjugates.

[0022] The isolated serotype 15A capsular saccharides obtained by purification of serotype 15A polysaccharide from S. pneumoniae lysate and, optionally, sizing of the purified polysaccharide, can be characterized by various parameters including, for example, weight average molecular weight (Mw).

[0023] The molecular weight of polysaccharides can be measured by size exclusion chromatography (SEC) coupled with a multi-angle laser light scattering detector (MALLS). In preferred embodiments, the isolated serotype 15A capsular polysaccharide (i.e., purified prior to further processing) has a weight average molecular weight of between 100 kDa and 2500 kDa. In one embodiment, the isolated serotype 15A capsular polysaccharide has a weight average molecular weight of between 250 kDa and 1500 kDa. In one embodiment, the isolated serotype 15A capsular polysaccharide has a weight average molecular weight of between 500 kDa and 1000 kDa.

[0024] Any integer within any of the above ranges is contemplated as an aspect of the present disclosure. To produce serotype 15A conjugates with advantageous filterability characteristics, immunogenicity, and / or yield, sizing of the polysaccharide to a target molecular weight range can be performed prior to conjugation to a carrier protein. Advantageously, the size of the purified serotype 15A polysaccharide is reduced while preserving important features of the polysaccharide's structure. Mechanical or chemical sizing can be used.

[0025] In one embodiment, the purified serotype 15A polysaccharide is reduced in size by chemical hydrolysis. Chemical hydrolysis can be carried out using a weak acid (e.g., acetic acid, formic acid, propanoic acid). Chemical hydrolysis can also be carried out using a dilute strong acid (e.g., dilute hydrochloric acid, dilute sulfuric acid, dilute phosphoric acid, dilute nitric acid, or dilute perchloric acid).

[0026] Preferably, however, the size of the purified serotype 15A polysaccharide is reduced by mechanical homogenization. In one embodiment, the size of the purified serotype 15A polysaccharide is reduced by high-pressure homogenization, which achieves high shear rates by pumping the process stream through channels of sufficiently small dimensions. The shear rate can be increased by using greater applied homogenization pressure, and the exposure time can be increased by recirculating the feed stream through the homogenizer.

[0027] In one embodiment, the isolated serotype 15A capsular polysaccharide is sized to a weight average molecular weight of between 50 kDa and 500 kDa. In a preferred embodiment, the isolated serotype 15A capsular polysaccharide is sized to a weight average molecular weight of between 75 kDa and 250 kDa. Preferably, the isolated serotype 15A capsular polysaccharide is sized to a weight average molecular weight of less than 175 kDa. In a more preferred embodiment, the isolated serotype 15A capsular polysaccharide is sized to a weight average molecular weight of between 75 kDa and 175 kDa. In a most preferred embodiment, the isolated serotype 15A capsular polysaccharide is sized to a weight average molecular weight of between 100 kDa and 175 kDa. Preferably, the isolated serotype 15A polysaccharide is sized by mechanical homogenization, preferably by high pressure homogenization.

[0028] In one aspect, the isolated serotype 15A capsular polysaccharide is unsized. In one embodiment, the isolated serotype 15A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 50 kDa and 500 kDa. In one embodiment, the isolated serotype 15A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 75 kDa and 250 kDa. In a preferred embodiment, the isolated serotype 15A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 75 kDa and 175 kDa. In a more preferred embodiment, the isolated serotype 15A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 90 kDa and 150 kDa. In a most preferred embodiment, the isolated serotype 15A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 175 kDa.

[0029] The weight average molecular weight (Mw) of a saccharide prior to conjugation refers to the Mw prior to activation of the polysaccharide (i.e., after the final sizing step, but before reacting the polysaccharide with an activating agent). In the context of the present invention, the Mw of the 15A polysaccharide is not substantially altered by the activation step, and the Mw of the 15A polysaccharide incorporated into the conjugate is similar to the Mw of the various polysaccharides measured prior to activation.

[0030] In one embodiment, the serotype 15A glycoconjugate of the invention comprises a serotype 15A capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 500 kDa. In one embodiment, the weight average molecular weight (Mw) is between 75 kDa and 250 kDa. In a preferred embodiment, the weight average molecular weight (Mw) is between 75 kDa and 175 kDa. In a most preferred embodiment, the weight average molecular weight (Mw) is between 90 kDa and 150 kDa.

[0031] In some embodiments, the serotype 15A glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 10,000 kDa. In other embodiments, the serotype 15A glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 10,000 kDa. Preferably, the serotype 15A glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 6,000 kDa.

[0032] The serotype 15A glycoconjugates of the present invention can also be characterized by the saccharide to carrier protein ratio (w / w). In some embodiments, the serotype 15A polysaccharide to carrier protein ratio (w / w) in the glycoconjugate is between 0.5 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.5 and 1.5. Even more preferably, the saccharide to carrier protein ratio (w / w) is between 0.7 and 1.1.

[0033] Another method for characterizing the serotype 15A glycoconjugates of the invention is by the number of lysine residues in the carrier protein (e.g., CRM197, DT, or TT) that become conjugated to a saccharide, which can be characterized as the extent of conjugated lysines (degree of conjugation). Evidence of lysine modification of the carrier protein by covalent attachment to a polysaccharide can be obtained by amino acid analysis using routine methods known to those of skill in the art. Conjugation results in a reduction in the number of recovered lysine residues compared to the carrier protein starting material used to generate the conjugate material. In preferred embodiments, the degree of conjugation of the serotype 15A glycoconjugates of the invention is between 2 and 20. Preferably, the degree of conjugation of the serotype 15A glycoconjugates of the invention is between 5 and 10.

[0034] Serotype 15A glycoconjugates and immunogenic compositions of the invention may contain free saccharides that are not covalently conjugated to a carrier protein but are nevertheless present in the glycoconjugate composition. The free saccharides may be non-covalently associated with (i.e., non-covalently bound to, adsorbed to, or entrapped in or with) the glycoconjugate.

[0035] In one embodiment, the serotype 15A glycoconjugate comprises less than about 40% free serotype 15A polysaccharide relative to the total amount of serotype 15A polysaccharide. In a preferred embodiment, the serotype 15A glycoconjugate comprises less than about 25% free serotype 15A polysaccharide relative to the total amount of serotype 15A polysaccharide.

[0036] Serotype 15A glycoconjugates can also be characterized by molecular size distribution (Kd). Size-exclusion chromatography media (CL-4B) can be used to determine the relative molecular size distribution of conjugates. Size-exclusion chromatography (SEC) is used in a gravity-fed column to profile the molecular size distribution of conjugates. Large molecules excluded from pores in the media elute more rapidly than small molecules. A fraction collector is used to collect the column eluate. Fractions are tested colorimetrically by saccharide assay. To determine Kd, the column is calibrated to establish the fraction at which molecules are completely excluded (V0), (Kd = 0), and the fraction representing maximum retention (Vi), (Kd = 1). The fraction at which a particular sample attribute is reached (Ve) is related to Kd by the expression Kd = (Ve - V0) / (Vi - V0).

[0037] In one embodiment, at least 40% of the serotype 15A glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 50% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 50% and 90% of the serotype 15A glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0038] In one aspect, the serotype 15A saccharide is activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide is then coupled, either directly or via a spacer (linker) group, to a carrier protein (preferably a CRMP). 197For example, the spacer can be cystamine or cysteamine, resulting in a thiolated polysaccharide, which can be coupled to the carrier by a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyrloxy] succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g., using iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl) aminobenzoate (SlAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA), or succinimidyl 3-[bromoacetamido] propionate (SBAP)). Preferably, the cyanate ester is coupled with hexanediamine or adipic acid dihydrazide (ADH), and the amino-derivatized saccharide is attached to the carrier protein (e.g., CRM) using carbodiimide (e.g., EDAC or EDC) chemistry via carboxyl groups on the protein carrier. 197 Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348 and WO 96 / 129094.

[0039] Other suitable techniques for conjugation use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU. Many are described in International Patent Application Publication No. WO 98 / 42721. Conjugation may involve a carbonyl linker, which can be formed by reaction of the free hydroxyl group of a sugar with 1,1'-carbonyldiimidazole (CDI) (see Bethell et al. (1979) J. Biol. Chern. 254:2572-2574; Hearn et al. (1981) J. Chromatogr. 218:509-518), followed by reaction with the protein to form a carbamate bond. This may involve reducing the anomeric terminus to a primary hydroxyl group, optionally protecting / deprotecting the primary hydroxyl group, reacting the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling the CDI carbamate intermediate to an amino group on the protein.

[0040] In a preferred embodiment, the serotype 15A glycoconjugates of the invention are prepared using reductive amination chemistry. According to the present invention, reductive amination involves two steps: (1) oxidizing (activating) purified saccharides and (2) reducing the activated saccharides and carrier protein to form the glycoconjugate (see, e.g., WO2006 / 110381, WO2008 / 079653, WO2008 / 143709, WO2008 / 079732, WO2011 / 110531, WO2012 / 119972, WO2015110941, WO2015110940, WO2018 / 144439, WO2018 / 156491).

[0041] In one aspect, the serotype 15A glycoconjugate of the invention is prepared by conjugating isolated serotype 15A capsular polysaccharide to a carrier protein by a process comprising the following steps: (a) reacting the isolated serotype 15A capsular polysaccharide with an oxidizing agent; (b) combining the activated polysaccharide of step (a) with a carrier protein; and (c) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate.

[0042] As noted above, sizing of the isolated serotype 15A capsular polysaccharide to a target molecular weight (MW) range may be performed prior to oxidation. Thus, in one aspect, the isolated serotype 15A capsular polysaccharide is sized prior to oxidation.

[0043] In one aspect, the isolated serotype 15A capsular polysaccharide is not sized prior to oxidation. In one aspect, the oxidation step (a) is carried out at a pH between 4.0 and 6.0. Preferably, the oxidation step (a) is carried out at a pH between 4.5 and 5.5.

[0044] In one aspect, the oxidation step (a) is carried out at a pH of about 5.0. The saccharides are said to be activated after the oxidation step (a) and are called "activated polysaccharides".

[0045] In one embodiment, the activated serotype 15A polysaccharide of the present invention has a weight average molecular weight (Mw) between 50 kDa and 500 kDa. In one embodiment, the weight average molecular weight (Mw) is between 75 kDa and 250 kDa. In a preferred embodiment, the weight average molecular weight (Mw) is between 75 kDa and 175 kDa. In a most preferred embodiment, the weight average molecular weight (Mw) is between 90 kDa and 150 kDa.

[0046] In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes terminal hydroxyl groups to aldehydes. In one embodiment, the oxidizing agent is periodate. For purposes of the present invention, the term "periodate" includes both periodate and periodic acid, and the term also includes metaperiodate (IO 4- ) and orthoperiodate (IO6 5-) and various salts of periodate (e.g., sodium periodate and potassium periodate).

[0047] In a preferred embodiment, the oxidizing agent is sodium periodate. In one embodiment, the periodate used for oxidation is metaperiodate. In a most preferred embodiment, the periodate used for oxidation is sodium metaperiodate.

[0048] When a polysaccharide is reacted with periodate, the periodate oxidizes vicinal hydroxyl groups to form carbonyl or aldehyde groups, causing cleavage of C-C bonds. For this reason, the term "reacting a polysaccharide with periodate" includes the oxidation of vicinal hydroxyl groups by periodate.

[0049] In one aspect, step a) comprises reacting the polysaccharide with 0.1 to 2 molar equivalents of periodate. Preferably, step a) comprises reacting the polysaccharide with 0.5 to 1.5 molar equivalents of periodate. Most preferably, step a) comprises reacting the polysaccharide with 0.8 to 1.2 molar equivalents of periodate.

[0050] In one embodiment, the degree of oxidation of the activated serotype 15A polysaccharide (also referred to herein as the "degree of activation") is between 2 and 20. In a preferred embodiment, the degree of oxidation of the activated serotype 15A polysaccharide is between 2 and 8. In a most preferred embodiment, the degree of oxidation of the activated serotype 15A polysaccharide is 5±2.5.

[0051] In one aspect, the activated serotype 15A polysaccharide and carrier protein are lyophilized prior to step b). Preferably, lyophilization occurs after step a). In one aspect, the activated polysaccharide is lyophilized after step a) and the carrier protein is also lyophilized.

[0052] In one embodiment, the activated serotype 15A polysaccharide is lyophilized after step a), the carrier protein is also lyophilized, and the activated polysaccharide and the carrier protein are reconstituted in the same solution.

[0053] In one embodiment, the activated serotype 15A polysaccharide and the carrier protein are lyophilized independently (separate lyophilization). In one embodiment, the activated serotype 15A polysaccharide and the carrier protein are lyophilized together (co-lyophilized).

[0054] In one aspect, lyophilization occurs in the presence of a non-reducing sugar, and possible non-reducing sugars include sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, and palatinit. In one aspect, the sugar is selected from the group consisting of sucrose, trehalose, and mannitol. In one aspect, the sugar is sucrose, trehalose, or mannitol. In one aspect, the sugar is sucrose.

[0055] In one aspect, the initial weight ratio of activated serotype 15A capsular polysaccharide to carrier protein in step b) is between 3:1 and 0.5:1. In one aspect, the initial weight ratio of activated serotype 15A capsular polysaccharide to carrier protein is between 1.5:1 and 0.5:1. Preferably, the initial weight ratio of activated serotype 15A capsular polysaccharide to carrier protein is between 1.1:1 and 0.9:1.

[0056] In one embodiment, the reduction reaction (c) is carried out in an aqueous solvent. Preferably, the reduction reaction (c) is carried out in an aprotic solvent. In one embodiment, the reduction reaction (c) is carried out in the presence of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). Preferably, the reduction reaction (c) is carried out in the presence of dimethyl sulfoxide (DMSO).

[0057] In one embodiment, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethyl formamide) solvent. Most preferably, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) solvent.

[0058] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium or zinc borohydride, zinc, or an amine borane, such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3, or 5-ethyl-2-methylpyridine borane (PEMB), in the presence of a Bronsted acid or Lewis acid. In one embodiment, the reducing agent is sodium triacetoxyborohydride. In one embodiment, the reducing agent is sodium cyanoborohydride in the presence of nickel (see WO2018144439). In a preferred embodiment, the reducing agent is sodium cyanoborohydride.

[0059] In one embodiment, between 0.2 and 5 molar equivalents of reducing agent are used in step c). Preferably, between 0.5 and 2 molar equivalents of reducing agent are used in step c). Most preferably, between 0.9 and 1.1 molar equivalents of reducing agent are used in step c).

[0060] At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugate, which can be capped using a suitable capping agent, which in one aspect is sodium borohydride (NaBH4).

[0061] In one aspect, capping is achieved by combining the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In one aspect, capping is achieved by combining the product of step c) with 1 to 3 molar equivalents of sodium borohydride.

[0062] After conjugation to a carrier protein, the serotype 15A glycoconjugate can be purified (concentrated with respect to the amount of saccharide-protein conjugate) by a variety of techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration operations, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. Thus, in one aspect, a process for producing a serotype 15A glycoconjugate of the invention includes a step of purifying the glycoconjugate after it is produced. 1.2 Streptococcus pneumoniae serotype 23A glycoconjugates of the present invention In one aspect, the present invention relates to Streptococcus pneumoniae serotype 23A glycoconjugates.

[0063] The structure of the Streptococcus pneumoniae serotype 23A polysaccharide is known in the art (see, e.g., Ravenscroft N et al. (2017) Carbohydrate Res. 450, 19-29 and WO2019050814). The structure of the serotype 23A capsular polysaccharide is: →4)-β-D-Glcp-(1→3)-[[α-L-Rhap-(1→2)]-[Gro-(2→P→3)]-β-D-Galp-(1→4)]-β-L-Rhap-(1→ is.

[0064] In one aspect the encapsulated S. pneumoniae serotype 23A saccharide used in the invention is a synthetic carbohydrate. However, in a preferred embodiment, the source of bacterial polysaccharide according to the present invention may be Streptococcus pneumoniae serotype 23A bacterial cells. Bacterial strains that can be used as a source of Streptococcus pneumoniae serotype 23A polysaccharide can be obtained from established culture collections (such as, for example, from the Streptococcus Reference Laboratory (US Centers for Disease Control and Prevention, Atlanta, GA, USA)) or from clinical specimens.

[0065] Serotype 23A saccharides can be obtained directly from bacteria using isolation procedures known to those skilled in the art, or they can be purchased commercially (e.g., from the American Type Culture Collection (ATCC), Manassas, Virginia, USA) (e.g., Reference No. ATCC 545-X, No. ATCC 546-X, No. ATCC 547-X).

[0066] When serotype 23A saccharides are obtained directly from bacteria, the bacterial cells can be grown in a medium, preferably a soy-based medium. After fermentation of the bacterial cells that produce Streptococcus pneumoniae serotype 23A capsular polysaccharide, the bacterial cells can be lysed to produce a cell lysate. The serotype 23A polysaccharide can then be isolated from the cell lysate using purification techniques known in the art, including centrifugation, depth filtration, precipitation, ultrafiltration, treatment with activated carbon, diafiltration, and / or column chromatography (see, e.g., US2006 / 0228380, US2006 / 0228381, WO2008 / 118752, and WO2020170190). The purified serotype 23A capsular polysaccharide can then be used to prepare glycoconjugates.

[0067] The isolated serotype 23A capsular saccharides obtained by purification of serotype 23A polysaccharide from S. pneumoniae lysate and, optionally, sizing of the purified polysaccharide, can be characterized by a variety of parameters including, for example, weight average molecular weight (Mw).

[0068] The molecular weight of polysaccharides can be measured by size exclusion chromatography (SEC) coupled with a multi-angle laser light scattering detector (MALLS). In preferred embodiments, the isolated serotype 23A capsular polysaccharide (i.e., purified prior to further processing) has a weight average molecular weight of between 100 kDa and 2500 kDa. In one embodiment, the isolated serotype 23A capsular polysaccharide has a weight average molecular weight of between 250 kDa and 1500 kDa. In one embodiment, the isolated serotype 23A capsular polysaccharide has a weight average molecular weight of between 500 kDa and 1000 kDa.

[0069] Any integer within any of the above ranges is contemplated as an aspect of the present disclosure. To produce serotype 23A conjugates with advantageous filterability characteristics, immunogenicity, and / or yield, sizing of the polysaccharide to a target molecular weight range can be performed prior to conjugation to a carrier protein. Advantageously, the size of the purified serotype 23A polysaccharide is reduced while retaining important features of the polysaccharide's structure. Mechanical or chemical sizing can be used.

[0070] Most preferably, the size of the purified serotype 23A polysaccharide is reduced by mechanical homogenization. For example, the use of acid hydrolysis as recommended in WO2019050814 or in WO2019050818 has been found to be inadequate for reducing the size of serotype 23A polysaccharide.

[0071] It has been found that acid hydrolysis can affect the structural integrity of serotype 23A polysaccharide: even relatively mild hydrolysis (e.g., 2 hours of treatment with 100 mM acetic acid at 80°C) has been found to lead to a 25% loss of branched rhamnose (α-L-Rhap-(1→2)) (see Figure 1).

[0072] Mechanical sizing can avoid the loss of this residue, which may be important from an immunological standpoint. Furthermore, this residue is the primary activation site for periodate oxidation. Therefore, it is important to preserve it when periodate oxidation of polysaccharides is used (e.g., as an activation step in reductive amination chemistry).

[0073] Therefore, in a preferred embodiment, the size of the purified serotype 23A polysaccharide is reduced by mechanical homogenization. In one embodiment, the size of purified serotype 23A polysaccharide is reduced by high-pressure homogenization, which achieves high shear rates by pumping the process stream through channels of sufficiently small dimensions. The shear rate is increased by using greater applied homogenization pressure, and the exposure time can be increased by recirculating the feed stream through the homogenizer.

[0074] In a most preferred embodiment, the process for preparing the serotype 23A glycoconjugates of the present invention does not include a step of sizing the serotype 23A polysaccharide by acid hydrolysis. In one embodiment, the isolated serotype 23A capsular polysaccharide is sized to a weight average molecular weight of between 50 kDa and 500 kDa. In a preferred embodiment, the isolated serotype 23A capsular polysaccharide is sized to a weight average molecular weight of between 75 kDa and 400 kDa. In a more preferred embodiment, the isolated serotype 23A capsular polysaccharide is sized to a weight average molecular weight of between 100 kDa and 350 kDa. In a most preferred embodiment, the isolated serotype 23A capsular polysaccharide is sized to a weight average molecular weight of between 125 kDa and 225 kDa. Preferably, the isolated serotype 23A polysaccharide is sized by mechanical homogenization, most preferably by high-pressure homogenization.

[0075] In one aspect, the isolated serotype 23A capsular polysaccharide is unsized. In one embodiment, the isolated serotype 23A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 50 kDa and 500 kDa. In one embodiment, the isolated serotype 23A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 75 kDa and 400 kDa. In a more preferred embodiment, the isolated serotype 23A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 350 kDa. In a most preferred embodiment, the isolated serotype 23A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 125 kDa and 225 kDa.

[0076] The weight average molecular weight (Mw) of a saccharide prior to conjugation refers to the Mw prior to activation of the polysaccharide (i.e., after the final sizing step, but before the polysaccharide is reacted with an activating agent). In the context of the present invention, the Mw of the 23A polysaccharide is not substantially altered by the activation step, and the Mw of the 23A polysaccharide incorporated into the conjugate is similar to the Mw of the various polysaccharides measured prior to activation.

[0077] In one embodiment, the serotype 23A glycoconjugate of the invention comprises a serotype 23A capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 400 kDa. In one embodiment, the weight average molecular weight (Mw) is between 100 kDa and 300 kDa. In a most preferred embodiment, the weight average molecular weight (Mw) is between 120 kDa and 240 kDa.

[0078] In some embodiments, the serotype 23A glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 10,000 kDa. In other embodiments, the serotype 23A glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 7,500 kDa. Preferably, the serotype 23A glycoconjugates have a weight average molecular weight (Mw) between 2,000 kDa and 5,000 kDa.

[0079] The serotype 23A glycoconjugates of the present invention can also be characterized by their saccharide to carrier protein ratio (w / w). In some embodiments, the serotype 23A polysaccharide to carrier protein ratio (w / w) in the glycoconjugate is between 0.5 and 3.0. Preferably, the saccharide to protein ratio (w / w) is between 0.7 and 1.5. Even more preferably, the saccharide to protein ratio (w / w) is between 0.8 and 1.4.

[0080] Another method for characterizing the serotype 23A glycoconjugates of the invention is by the number of lysine residues in the carrier protein (e.g., CRM197, DT, or TT) that become conjugated to a saccharide, which can be characterized as the extent of conjugated lysines (degree of conjugation). Evidence of lysine modification of the carrier protein by covalent attachment to a polysaccharide can be obtained by amino acid analysis using routine methods known to those skilled in the art. Conjugation results in a reduction in the number of recovered lysine residues compared to the carrier protein starting material used to generate the conjugate material. In preferred embodiments, the degree of conjugation of the serotype 23A glycoconjugates of the invention is between 2 and 20. Preferably, the degree of conjugation of the serotype 23A glycoconjugates of the invention is between 5 and 15.

[0081] Serotype 23A glycoconjugates and immunogenic compositions of the invention may contain free saccharides that are not covalently conjugated to a carrier protein but are nevertheless present in the glycoconjugate composition. The free saccharides may be non-covalently associated with the glycoconjugate (i.e., non-covalently bound to, adsorbed to, or entrapped in or with it).

[0082] In one embodiment, the serotype 23A glycoconjugate comprises less than about 40% free serotype 23A polysaccharide relative to the total amount of serotype 23A polysaccharide. In a preferred embodiment, the serotype 23A glycoconjugate comprises less than about 25% free serotype 23A polysaccharide relative to the total amount of serotype 23A polysaccharide.

[0083] Serotype 23A glycoconjugates can also be characterized by molecular size distribution (Kd). Size-exclusion chromatography media (CL-4B) can be used to determine the relative molecular size distribution of conjugates. Size-exclusion chromatography (SEC) is used in a gravity-fed column to profile the molecular size distribution of conjugates. Large molecules excluded from pores in the media elute more rapidly than small molecules. A fraction collector is used to collect the column eluate. Fractions are tested colorimetrically by saccharide assay. To determine Kd, the column is calibrated to establish the fraction at which molecules are completely excluded (V0), (Kd = 0), and the fraction representing maximum retention (Vi), (Kd = 1). The fraction at which a particular sample attribute is reached (Ve) is related to Kd by the expression Kd = (Ve - V0) / (Vi - V0).

[0084] In one embodiment, at least 40% of the serotype 23A glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 50% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 50% and 90% of the serotype 23A glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0085] Serotype 23A glycoconjugates can also be characterized by the amount of branching rhamnose residues remaining in the 23A polysaccharide. As noted above, it has been found that serotype 23A polysaccharide can lose branching rhamnose residues (see Figure 1).

[0086] Thus, in one aspect, a Streptococcus pneumoniae serotype 23A glycoconjugate of the invention comprises a Streptococcus pneumoniae serotype 23A capsular polysaccharide, which has a branched rhamnose content of greater than 75% compared to native Streptococcus pneumoniae serotype 23A capsular polysaccharide, which is believed to have a branched rhamnose content of about 100%. In one aspect, a Streptococcus pneumoniae serotype 23A glycoconjugate of the invention comprises a Streptococcus pneumoniae serotype 23A capsular polysaccharide, which has a branched rhamnose content of greater than 90% compared to native Streptococcus pneumoniae serotype 23A capsular polysaccharide, which is believed to have a branched rhamnose content of about 100%. Preferably, the Streptococcus pneumoniae serotype 23A glycoconjugate of the invention comprises a Streptococcus pneumoniae serotype 23A capsular polysaccharide, said Streptococcus pneumoniae serotype 23A capsular polysaccharide having a branched rhamnose content of greater than 95% compared to native Streptococcus pneumoniae serotype 23A capsular polysaccharide, which is believed to have a branched rhamnose content of about 100%.

[0087] In a most preferred embodiment, the Streptococcus pneumoniae serotype 23A glycoconjugate of the invention comprises a Streptococcus pneumoniae serotype 23A capsular polysaccharide, said Streptococcus pneumoniae serotype 23A capsular polysaccharide having a branched rhamnose content of about 100% compared to native Streptococcus pneumoniae serotype 23A capsular polysaccharide, which is believed to have a branched rhamnose content of about 100%.

[0088] In one aspect, the serotype 23A saccharide is activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide is then coupled, either directly or via a spacer (linker) group, to a carrier protein (preferably a CRMP). 197For example, the spacer can be cystamine or cysteamine, resulting in a thiolated polysaccharide, which can be coupled to the carrier by a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyrloxy] succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g., using iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl) aminobenzoate (SlAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA), or succinimidyl 3-[bromoacetamido] propionate (SBAP)). Preferably, the cyanate ester is coupled with hexanediamine or adipic acid dihydrazide (ADH), and the amino-derivatized saccharide is attached to the carrier protein (e.g., CRM) using carbodiimide (e.g., EDAC or EDC) chemistry via carboxyl groups on the protein carrier. 197 Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348 and WO 96 / 129094.

[0089] Other suitable techniques for conjugation use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU. Many are described in International Patent Application Publication No. WO 98 / 42721. Conjugation may involve a carbonyl linker, which can be formed by reaction of the free hydroxyl group of a sugar with 1,1'-carbonyldiimidazole (CDI) (see Bethell et al. (1979) J. Biol. Chern. 254:2572-2574; Hearn et al. (1981) J. Chromatogr. 218:509-518), followed by reaction with the protein to form a carbamate bond. This may involve reducing the anomeric terminus to a primary hydroxyl group, optionally protecting / deprotecting the primary hydroxyl group, reacting the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling the CDI carbamate intermediate to an amino group on the protein.

[0090] In a preferred embodiment, the serotype 23A glycoconjugates of the invention are prepared using reductive amination chemistry. According to the present invention, reductive amination involves two steps: (1) oxidizing (activating) purified saccharides, and (2) reducing the activated saccharides and carrier protein to form the glycoconjugate (see, e.g., WO2006 / 110381, WO2008 / 079653, WO2008 / 143709, WO2008 / 079732, WO2011 / 110531, WO2012 / 119972, WO2015110941, WO2015110940, WO2018 / 144439, WO2018 / 156491).

[0091] In one aspect, the serotype 23A glycoconjugate of the invention is prepared by conjugating isolated serotype 23A capsular polysaccharide to a carrier protein by a process comprising the following steps: (a) reacting the isolated serotype 23A capsular polysaccharide with an oxidizing agent; (b) combining the activated polysaccharide of step (a) with a carrier protein; and (c) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate.

[0092] As noted above, sizing of the isolated serotype 23A capsular polysaccharide to a target molecular weight (MW) range may be performed prior to oxidation. Thus, in one aspect, the isolated serotype 23A capsular polysaccharide is sized prior to oxidation.

[0093] In a preferred embodiment, the isolated serotype 23A capsular polysaccharide is size reduced by mechanical homogenization. In one embodiment, the isolated serotype 23A polysaccharide is size reduced by high-pressure homogenization. In a most preferred embodiment, the isolated serotype 23A capsular polysaccharide is not sized by acid hydrolysis.

[0094] In one embodiment, the isolated serotype 23A capsular polysaccharide is not sized prior to oxidation. In one aspect, the oxidation step (a) is carried out at a pH between 4.5 and 6.5. Preferably, the oxidation step (a) is carried out at a pH between 5.0 and 6.0.

[0095] In one aspect, the oxidation step (a) is carried out at a pH of about 5.0. The saccharides are said to be activated after the oxidation step (a) and are called "activated polysaccharides".

[0096] In one embodiment, the activated serotype 23A polysaccharide of the present invention has a weight average molecular weight (Mw) between 50 kDa and 400 kDa. In one embodiment, the weight average molecular weight (Mw) is between 100 kDa and 250 kDa. In a most preferred embodiment, the weight average molecular weight (Mw) is between 120 kDa and 200 kDa.

[0097] In one embodiment, the activated serotype 23A polysaccharide of the present invention retains at least 80% branched rhamnose. In one embodiment, the activated serotype 23A polysaccharide of the present invention retains at least 85% branched rhamnose. In one embodiment, the activated serotype 23A polysaccharide of the present invention retains at least 90% branched rhamnose. In a preferred embodiment, the activated serotype 23A polysaccharide of the present invention retains at least 95% branched rhamnose.

[0098] In a preferred embodiment, the activated serotype 23A polysaccharide of the present invention retains at least 96% branched rhamnose. In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes terminal hydroxyl groups to aldehydes. In one embodiment, the oxidizing agent is periodate. For purposes of the present invention, the term "periodate" includes both periodate and periodic acid, and the term also includes metaperiodate (IO 4- ) and orthoperiodate (IO6 5- ) and various salts of periodate (e.g., sodium periodate and potassium periodate).

[0099] In a preferred embodiment, the oxidizing agent is sodium periodate. In one embodiment, the periodate used for oxidation is metaperiodate. In a most preferred embodiment, the periodate used for oxidation is sodium metaperiodate.

[0100] When a polysaccharide is reacted with periodate, the periodate oxidizes vicinal hydroxyl groups to form carbonyl or aldehyde groups, causing cleavage of C-C bonds. For this reason, the term "reacting a polysaccharide with periodate" includes the oxidation of vicinal hydroxyl groups by periodate.

[0101] In one aspect, step a) comprises reacting the polysaccharide with 0.1 to 2 molar equivalents of periodate. Preferably, step a) comprises reacting the polysaccharide with 0.2 to 1.5 molar equivalents of periodate. Most preferably, step a) comprises reacting the polysaccharide with 0.3 to 0.5 molar equivalents of periodate.

[0102] In one embodiment, the degree of oxidation of the activated serotype 23A polysaccharide (also referred to herein as the "degree of activation") is between 2 and 20. In a preferred embodiment, the degree of oxidation of the activated serotype 23A polysaccharide is between 2 and 8. In a most preferred embodiment, the degree of oxidation of the activated serotype 23A polysaccharide is 5±2.5.

[0103] In one aspect, the activated serotype 23A polysaccharide and carrier protein are lyophilized prior to step b). Preferably, lyophilization occurs after step a). In one aspect, the activated polysaccharide is lyophilized after step a) and the carrier protein is also lyophilized. In one aspect, the activated serotype 23A polysaccharide is lyophilized after step a) and the carrier protein is also lyophilized, and the activated polysaccharide and carrier protein are reconstituted in the same solution.

[0104] In one embodiment, the activated serotype 23A polysaccharide and the carrier protein are lyophilized independently (separate lyophilization). In one embodiment, the activated serotype 23A polysaccharide and the carrier protein are lyophilized together (co-lyophilized).

[0105] In one aspect, lyophilization occurs in the presence of a non-reducing sugar, and possible non-reducing sugars include sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, and palatinit. In one aspect, the sugar is selected from the group consisting of sucrose, trehalose, and mannitol. In one aspect, the sugar is sucrose, trehalose, or mannitol. In one aspect, the sugar is sucrose.

[0106] In one aspect, the initial weight ratio of activated serotype 23A capsular polysaccharide to carrier protein in step b) is between 2:1 and 0.5:1. In one aspect, the initial weight ratio of activated serotype 23A capsular polysaccharide to carrier protein is between 1.2:1 and 0.6:1. Preferably, the initial weight ratio of activated serotype 23A capsular polysaccharide to carrier protein is between 0.9:1 and 0.7:1.

[0107] In one embodiment, the reduction reaction (c) is carried out in an aqueous solvent. Preferably, the reduction reaction (c) is carried out in an aprotic solvent. In one embodiment, the reduction reaction (c) is carried out in the presence of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). Preferably, the reduction reaction (c) is carried out in the presence of dimethyl sulfoxide (DMSO).

[0108] In one embodiment, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethyl formamide) solvent. Most preferably, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) solvent.

[0109] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium or zinc borohydride, amine borane, such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane (PEMB) in the presence of Bronsted acid or Lewis acid. In one embodiment, the reducing agent is sodium cyanoborohydride in the presence of nickel (see WO2018144439). In a preferred embodiment, the reducing agent is sodium cyanoborohydride.

[0110] In one embodiment, between 0.2 and 5 molar equivalents of reducing agent are used in step c). Preferably, between 0.2 and 1.5 molar equivalents of reducing agent are used in step c). Most preferably, between 0.5 and 1.0 molar equivalents of reducing agent are used in step c).

[0111] At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugate, which can be capped using a suitable capping agent, which in one aspect is sodium borohydride (NaBH4).

[0112] In one aspect, capping is achieved by combining the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In one aspect, capping is achieved by combining the product of step c) with 1 to 3 molar equivalents of sodium borohydride.

[0113] After conjugation to a carrier protein, the serotype 23A glycoconjugate can be purified (concentrated with respect to the amount of saccharide-protein conjugate) by a variety of techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration operations, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. Thus, in one aspect, a process for producing a serotype 235A glycoconjugate of the invention includes a step of purifying the glycoconjugate after it has been produced. 1.3 Streptococcus pneumoniae serotype 23B glycoconjugates of the present invention In one aspect, the present invention relates to Streptococcus pneumoniae serotype 23B glycoconjugates.

[0114] The structure of the Streptococcus pneumoniae serotype 23B polysaccharide is known in the art (see, e.g., Ravenscroft N et al. (2017) Carbohydrate Res. 450, 19-29 and WO2019050814). The structure of the serotype 23A capsular polysaccharide is: →4)-β-D-Glcp-(1→4)-[Gro-(2→P→3)]-β-D-Galp-(1→4)-β-L-Rhap-(1→ is.

[0115] In one aspect the encapsulated S. pneumoniae serotype 23B saccharide used in the invention is a synthetic carbohydrate. However, in a preferred embodiment, the source of bacterial polysaccharide according to the present invention may be Streptococcus pneumoniae serotype 23B bacterial cells. Bacterial strains that can be used as a source of Streptococcus pneumoniae serotype 23B polysaccharide can be obtained from established culture collections (such as, for example, from the Streptococcus Reference Laboratory (US Centers for Disease Control and Prevention, Atlanta, GA, USA)) or from clinical specimens.

[0116] Serotype 23B saccharides can be obtained directly from bacteria using isolation procedures known to those skilled in the art, or they can be purchased commercially (e.g., from the American Type Culture Collection (ATCC), Manassas, Virginia, USA) (e.g., Reference No. ATCC 548-X, No. ATCC 549-X, No. ATCC 550-X).

[0117] When serotype 23B saccharides are obtained directly from bacteria, the bacterial cells can be grown in a medium, preferably a soy-based medium. After fermentation of the bacterial cells that produce Streptococcus pneumoniae serotype 23B capsular polysaccharide, the bacterial cells can be lysed to produce a cell lysate. The serotype 23B polysaccharide can then be isolated from the cell lysate using purification techniques known in the art, including centrifugation, depth filtration, precipitation, ultrafiltration, treatment with activated carbon, diafiltration, and / or column chromatography (see, e.g., US2006 / 0228380, US2006 / 0228381, WO2008 / 118752, and WO2020170190). The purified serotype 23B capsular polysaccharide can be used to prepare glycoconjugates.

[0118] The isolated serotype 23B capsular saccharides obtained by purification of serotype 23B polysaccharide from S. pneumoniae lysate and, optionally, sizing of the purified polysaccharide, can be characterized by various parameters including, for example, weight average molecular weight (Mw).

[0119] The molecular weight of polysaccharides can be measured by size exclusion chromatography (SEC) coupled with a multi-angle laser light scattering detector (MALLS). In preferred embodiments, the isolated serotype 23B capsular polysaccharide (i.e., purified prior to further processing) has a weight average molecular weight of between 100 kDa and 2500 kDa. In one embodiment, the isolated serotype 23B capsular polysaccharide has a weight average molecular weight of between 250 kDa and 2000 kDa. In one embodiment, the isolated serotype 23B capsular polysaccharide has a weight average molecular weight of between 300 kDa and 1000 kDa.

[0120] Any integer within any of the above ranges is contemplated as an aspect of the present disclosure. To produce serotype 23B conjugates with advantageous filterability characteristics, immunogenicity, and / or yield, sizing of the polysaccharide to a target molecular weight range can be performed prior to conjugation to a carrier protein. Advantageously, the size of the purified serotype 23B polysaccharide is reduced while preserving important features of the polysaccharide's structure. Mechanical or chemical sizing can be used.

[0121] In one embodiment, the size of the purified serotype 23B polysaccharide is reduced by chemical hydrolysis. Chemical hydrolysis can be carried out using a weak acid (e.g., acetic acid, formic acid, propanoic acid). Chemical hydrolysis can also be carried out using a dilute strong acid (e.g., dilute hydrochloric acid, dilute sulfuric acid, dilute phosphoric acid, dilute nitric acid, or dilute perchloric acid).

[0122] Preferably, however, the size of the purified serotype 23B polysaccharide is reduced by mechanical homogenization. In one embodiment, the size of the purified serotype 23B polysaccharide is reduced by high-pressure homogenization, which achieves a high shear rate by pumping the process stream through a channel of sufficiently small dimensions. The shear rate can be increased by using a greater applied homogenization pressure, and the exposure time can be increased by recirculating the feed stream through the homogenizer.

[0123] In one embodiment, the isolated serotype 23B capsular polysaccharide is sized to a weight average molecular weight of between 50 kDa and 750 kDa. In a preferred embodiment, the isolated serotype 23B capsular polysaccharide is sized to a weight average molecular weight of between 75 kDa and 400 kDa. In an even more preferred embodiment, the isolated serotype 23B capsular polysaccharide is sized to a weight average molecular weight of between 100 kDa and 250 kDa. Preferably, the isolated serotype 23B polysaccharide is sized by mechanical homogenization, preferably by high pressure homogenization.

[0124] In one aspect, the isolated serotype 23B capsular polysaccharide is unsized. In one embodiment, the isolated serotype 23B capsular polysaccharide prior to conjugation has a weight average molecular weight of between 50 kDa and 750 kDa. In one embodiment, the isolated serotype 23B capsular polysaccharide prior to conjugation has a weight average molecular weight of between 75 kDa and 400 kDa. In a more preferred embodiment, the isolated serotype 23B capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 250 kDa.

[0125] The weight average molecular weight (Mw) of a saccharide prior to conjugation refers to the Mw prior to activation of the polysaccharide (i.e., after the final sizing step, but before reacting the polysaccharide with an activating agent). In the context of the present invention, the Mw of the 23B polysaccharide is not substantially altered by the activation step, and the Mw of the 23B polysaccharide incorporated into the conjugate is similar to the Mw of the various polysaccharides measured prior to activation.

[0126] In one embodiment, the serotype 23B glycoconjugate of the invention comprises a serotype 23B capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 40 kDa and 600 kDa. In one embodiment, the weight average molecular weight (Mw) is between 50 kDa and 300 kDa. In a most preferred embodiment, the weight average molecular weight (Mw) is between 100 kDa and 200 kDa.

[0127] In some embodiments, the serotype 23B glycoconjugates of the present invention have a weight average molecular weight (Mw) between 250 kDa and 7,500 kDa. In other embodiments, the serotype 23B glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 4,000 kDa. Preferably, the serotype 23B glycoconjugates of the present invention have a weight average molecular weight (Mw) between 700 kDa and 2,000 kDa.

[0128] The serotype 23B glycoconjugates of the present invention can also be characterized by the saccharide to carrier protein ratio (w / w). In some embodiments, the serotype 15A polysaccharide to carrier protein ratio (w / w) in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.5 and 1.5. Even more preferably, the saccharide to carrier protein ratio (w / w) is between 0.6 and 1.3.

[0129] Another method for characterizing the serotype 23B glycoconjugates of the present invention is by the number of lysine residues in the carrier protein (e.g., CRM197, DT, or TT) that become conjugated to a saccharide, which can be characterized as the extent of conjugated lysines (degree of conjugation). Evidence of lysine modification of the carrier protein by covalent attachment to a polysaccharide can be obtained by amino acid analysis using routine methods known to those of skill in the art. Conjugation results in a reduction in the number of recovered lysine residues compared to the carrier protein starting material used to generate the conjugate material. In preferred embodiments, the degree of conjugation of the serotype 23B glycoconjugates of the present invention is between 2 and 15. Preferably, the degree of conjugation of the serotype 23B glycoconjugates of the present invention is between 5 and 12.

[0130] Serotype 23B glycoconjugates and immunogenic compositions of the invention may contain free saccharides that are not covalently conjugated to a carrier protein but are nevertheless present in the glycoconjugate composition. The free saccharides may be non-covalently associated with the glycoconjugate (i.e., non-covalently bound to, adsorbed to, or entrapped in or with it).

[0131] In one embodiment, the serotype 23B glycoconjugate comprises less than about 40% free serotype 23B polysaccharide relative to the total amount of serotype 23B polysaccharide. In a preferred embodiment, the serotype 23B glycoconjugate comprises less than about 25% free serotype 23B polysaccharide relative to the total amount of serotype 23B polysaccharide.

[0132] Serotype 23B glycoconjugates can be characterized by molecular size distribution (Kd). Size-exclusion chromatography media (CL-4B) can be used to determine the relative molecular size distribution of conjugates. Size-exclusion chromatography (SEC) is used in a gravity-fed column to profile the molecular size distribution of conjugates. Large molecules excluded from pores in the media elute more rapidly than small molecules. A fraction collector is used to collect the column eluate. Fractions are tested colorimetrically by saccharide assay. To determine Kd, the column is calibrated to establish the fraction at which molecules are completely excluded (V0), (Kd = 0), and the fraction representing maximum retention (Vi), (Kd = 1). The fraction at which a particular sample attribute is reached (Ve) is related to Kd by the expression Kd = (Ve - V0) / (Vi - V0).

[0133] In one embodiment, at least 30% of the serotype 23B glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 35% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 40% and 60% of the serotype 23B glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0134] In one aspect, serotype 23B saccharides are activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form cyanate esters. The activated polysaccharides are then coupled, either directly or via a spacer (linker) group, to a carrier protein (preferably a CRMP). 197For example, the spacer can be cystamine or cysteamine, resulting in a thiolated polysaccharide, which can be coupled to the carrier by a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyrloxy] succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g., using iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl) aminobenzoate (SlAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA), or succinimidyl 3-[bromoacetamido] propionate (SBAP)). Preferably, the cyanate ester is coupled with hexanediamine or adipic acid dihydrazide (ADH), and the amino-derivatized saccharide is attached to the carrier protein (e.g., CRM) using carbodiimide (e.g., EDAC or EDC) chemistry via carboxyl groups on the protein carrier. 197 Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348 and WO 96 / 129094.

[0135] Other suitable techniques for conjugation use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU. Many are described in International Patent Application Publication No. WO 98 / 42721. Conjugation may involve a carbonyl linker, which can be formed by reaction of the free hydroxyl group of a sugar with 1,1'-carbonyldiimidazole (CDI) (see Bethell et al. (1979) J. Biol. Chern. 254:2572-2574; Hearn et al. (1981) J. Chromatogr. 218:509-518), followed by reaction with the protein to form a carbamate bond. This may involve reducing the anomeric terminus to a primary hydroxyl group, optionally protecting / deprotecting the primary hydroxyl group, reacting the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling the CDI carbamate intermediate to an amino group on the protein.

[0136] In a preferred embodiment, the serotype 23B glycoconjugates of the invention are prepared using reductive amination chemistry. According to the present invention, reductive amination involves two steps: (1) oxidizing (activating) purified saccharides and (2) reducing the activated saccharides and carrier protein to form the glycoconjugate (see, e.g., WO2006 / 110381, WO2008 / 079653, WO2008 / 143709, WO2008 / 079732, WO2011 / 110531, WO2012 / 119972, WO2015110941, WO2015110940, WO2018 / 144439, WO2018 / 156491).

[0137] In one aspect, the serotype 23B glycoconjugate of the invention is prepared by conjugating isolated serotype 23B capsular polysaccharide to a carrier protein by a process comprising the following steps: (a) reacting the isolated serotype 23B capsular polysaccharide with an oxidizing agent; (b) combining the activated polysaccharide of step (a) with a carrier protein; and (c) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate.

[0138] As noted above, sizing of the isolated serotype 23B capsular polysaccharide to a target molecular weight (MW) range may be performed prior to oxidation. Thus, in one aspect, the isolated serotype 23B capsular polysaccharide is sized prior to oxidation.

[0139] In a preferred embodiment, the size of the isolated serotype 23B is reduced by mechanical homogenization of the capsular polysaccharide. In one embodiment, the size of the isolated serotype 23B polysaccharide is reduced by high pressure homogenization.

[0140] In one aspect, the isolated serotype 23B capsular polysaccharide is not sized prior to oxidation. In one aspect, the oxidation step (a) is carried out at a pH between 4.0 and 6.5. Preferably, the oxidation step (a) is carried out at a pH between 4.5 and 5.5.

[0141] In one aspect, the oxidation step (a) is carried out at a pH of about 5.0. The saccharides are said to be activated after the oxidation step (a) and are called "activated polysaccharides".

[0142] In one embodiment, the activated serotype 23B polysaccharide of the present invention has a weight average molecular weight (Mw) between 40 kDa and 600 kDa. In one embodiment, the weight average molecular weight (Mw) is between 50 kDa and 300 kDa. In a most preferred embodiment, the weight average molecular weight (Mw) is between 100 kDa and 200 kDa.

[0143] In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes terminal hydroxyl groups to aldehydes. In one embodiment, the oxidizing agent is periodate. For purposes of the present invention, the term "periodate" includes both periodate and periodic acid, and the term also includes metaperiodate (IO4- ) and orthoperiodate (IO6 5- ) and various salts of periodate (e.g., sodium periodate and potassium periodate).

[0144] In a preferred embodiment, the oxidizing agent is sodium periodate. In one embodiment, the periodate used for oxidation is metaperiodate. In a most preferred embodiment, the periodate used for oxidation is sodium metaperiodate.

[0145] When a polysaccharide is reacted with periodate, the periodate oxidizes vicinal hydroxyl groups to form carbonyl or aldehyde groups, causing cleavage of C-C bonds. For this reason, the term "reacting a polysaccharide with periodate" includes the oxidation of vicinal hydroxyl groups by periodate.

[0146] In one aspect, step a) comprises reacting the polysaccharide with 0.05 to 1 molar equivalent of periodate. Preferably, step a) comprises reacting the polysaccharide with 0.1 to 0.3 molar equivalent of periodate. Most preferably, step a) comprises reacting the polysaccharide with about 0.2 molar equivalent of periodate.

[0147] In one embodiment, the degree of oxidation of the activated serotype 23B polysaccharide (also referred to herein as the "degree of activation") is between 2 and 20. In a preferred embodiment, the degree of oxidation of the activated serotype 23B polysaccharide is between 4 and 15. In a most preferred embodiment, the degree of oxidation of the activated serotype 23B polysaccharide is 9±3.

[0148] In one aspect, the activated serotype 23B polysaccharide and carrier protein are lyophilized prior to step b). Preferably, lyophilization occurs after step a). In one aspect, the activated polysaccharide is lyophilized after step a) and the carrier protein is also lyophilized. In one aspect, the activated serotype 23B polysaccharide is lyophilized after step a) and the carrier protein is also lyophilized, and the activated polysaccharide and carrier protein are reconstituted in the same solution.

[0149] In one embodiment, the activated serotype 23B polysaccharide and the carrier protein are lyophilized independently (separate lyophilization). In one embodiment, the activated serotype 23B polysaccharide and the carrier protein are lyophilized together (co-lyophilized).

[0150] In one aspect, lyophilization occurs in the presence of a non-reducing sugar, and possible non-reducing sugars include sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, and palatinit. In one aspect, the sugar is selected from the group consisting of sucrose, trehalose, and mannitol. In one aspect, the sugar is sucrose, trehalose, or mannitol. In one aspect, the sugar is sucrose.

[0151] In one aspect, the initial weight ratio of activated serotype 23B capsular polysaccharide to carrier protein in step b) is between 2:1 and 0.5:1. In one aspect, the initial weight ratio of activated serotype 23B capsular polysaccharide to carrier protein is between 1.2:1 and 0.6:1. Preferably, the initial weight ratio of activated serotype 23B capsular polysaccharide to carrier protein is between 0.9:1 and 0.7:1.

[0152] In one embodiment, the reduction reaction (c) is carried out in an aqueous solvent. Preferably, the reduction reaction (c) is carried out in an aprotic solvent. In one embodiment, the reduction reaction (c) is carried out in the presence of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). Preferably, the reduction reaction (c) is carried out in the presence of dimethyl sulfoxide (DMSO).

[0153] In one embodiment, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethyl formamide) solvent. Most preferably, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) solvent.

[0154] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium or zinc borohydride, amine borane, such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3, or 5-ethyl-2-methylpyridine borane (PEMB), in the presence of a Bronsted acid or Lewis acid. In one embodiment, the reducing agent is sodium triacetoxyborohydride. In one embodiment, the reducing agent is sodium cyanoborohydride in the presence of nickel (see WO2018144439). In a preferred embodiment, the reducing agent is sodium cyanoborohydride.

[0155] In one embodiment, between 0.2 and 5 molar equivalents of reducing agent are used in step c). Preferably, between 0.5 and 1.5 molar equivalents of reducing agent are used in step c). Most preferably, between 0.9 and 1.1 molar equivalents of reducing agent are used in step c).

[0156] At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugate, which can be capped using a suitable capping agent, which in one aspect is sodium borohydride (NaBH4).

[0157] In one aspect, capping is achieved by combining the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In one aspect, capping is achieved by combining the product of step c) with 1 to 3 molar equivalents of sodium borohydride.

[0158] After conjugation to a carrier protein, the serotype 23B glycoconjugate can be purified (concentrated with respect to the amount of saccharide-protein conjugate) by a variety of techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration operations, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. Thus, in one aspect, a process for producing a serotype 23B glycoconjugate of the invention includes a step of purifying the glycoconjugate after it has been produced. 1.4 Streptococcus pneumoniae serotype 24F glycoconjugates of the present invention In one aspect, the present invention relates to Streptococcus pneumoniae serotype 24F glycoconjugates.

[0159] The structure of the Streptococcus pneumoniae serotype 24F polysaccharide is known in the art (see, for example, WO2019050815). In one aspect the encapsulated S. pneumoniae serotype 24F saccharide used in the invention is a synthetic carbohydrate.

[0160] However, in a preferred embodiment, the source of bacterial polysaccharide according to the present invention may be Streptococcus pneumoniae serotype 24F bacterial cells. Bacterial strains that can be used as a source of Streptococcus pneumoniae serotype 24F polysaccharide can be obtained from established culture collections (such as, for example, from the Streptococcus Reference Laboratory (US Centers for Disease Control and Prevention, Atlanta, GA, USA)) or from clinical specimens.

[0161] Serotype 24F saccharides can be obtained directly from bacteria using isolation procedures known to those skilled in the art, or they can be purchased commercially (e.g., from the American Type Culture Collection (ATCC), Manassas, Virginia, USA) (e.g., Reference No. ATCC 551-X, No. ATCC 552-X, No. ATCC 553-X).

[0162] When serotype 24F saccharides are obtained directly from bacteria, the bacterial cells can be grown in a medium, preferably a soy-based medium. After fermentation of the bacterial cells that produce Streptococcus pneumoniae serotype 24F capsular polysaccharide, the bacterial cells can be lysed to produce a cell lysate. The serotype 24F polysaccharide can then be isolated from the cell lysate using purification techniques known in the art, including centrifugation, depth filtration, precipitation, ultrafiltration, treatment with activated carbon, diafiltration, and / or column chromatography (see, e.g., US2006 / 0228380, US2006 / 0228381, WO2008 / 118752, and WO2020170190). The purified serotype 24F capsular polysaccharide can then be used to prepare glycoconjugates.

[0163] The isolated serotype 24F capsular saccharides obtained by purification of serotype 24F polysaccharide from S. pneumoniae lysate and, optionally, sizing of the purified polysaccharide, can be characterized by various parameters including, for example, weight average molecular weight (Mw).

[0164] The molecular weight of polysaccharides can be measured by size exclusion chromatography (SEC) coupled with a multi-angle laser light scattering detector (MALLS). In preferred embodiments, the isolated serotype 24F capsular polysaccharide (i.e., purified prior to further processing) has a weight average molecular weight of between 100 kDa and 2500 kDa. In one embodiment, the isolated serotype 24F capsular polysaccharide has a weight average molecular weight of between 250 kDa and 2000 kDa. In one embodiment, the isolated serotype 24F capsular polysaccharide has a weight average molecular weight of between 500 kDa and 1000 kDa.

[0165] Any integer within any of the above ranges is contemplated as an aspect of the present disclosure. To produce serotype 24F conjugates with advantageous filterability characteristics, immunogenicity, and / or yield, sizing of the polysaccharide to a target molecular weight range can be performed prior to conjugation to a carrier protein. Advantageously, the size of the purified serotype 24F polysaccharide is reduced while retaining important structural features of the polysaccharide. Mechanical or chemical sizing can be used.

[0166] Most preferably, the size of the purified serotype 24F polysaccharide is reduced by mechanical homogenization. For example, the use of acid hydrolysis as recommended in WO2019050815 or in WO2019050818 has been found to be inadequate for reducing the size of serotype 24F polysaccharide.

[0167] It has been found that acid hydrolysis can affect the structural integrity of serotype 24F polysaccharide: even relatively mild hydrolysis (e.g., 2 hours of treatment with 100 mM acetic acid at 80°C) has been found to lead to a 25% loss of branched ribose residues (see Figure 2).

[0168] Mechanical sizing may avoid the loss of this residue, which may be important from an immunological point of view. Therefore, in a preferred embodiment, the size of purified serotype 24F is reduced by polysaccharide mechanical homogenization.

[0169] In one embodiment, the size of purified serotype 24F polysaccharide is reduced by high-pressure homogenization. High shear rates are achieved by pumping the high-pressure homogenization process stream through a channel of sufficiently small dimensions. The shear rate is increased by using greater applied homogenization pressure, and exposure time can be increased by recirculating the feed stream through the homogenizer.

[0170] In a most preferred embodiment, the process for preparing the serotype 24F glycoconjugates of the present invention does not include a step of sizing the serotype 24F polysaccharide by acid hydrolysis. In one embodiment, the isolated serotype 24F capsular polysaccharide is sized to a weight average molecular weight of between 50 kDa and 500 kDa. In a preferred embodiment, the isolated serotype 24F capsular polysaccharide is sized to a weight average molecular weight of between 75 kDa and 400 kDa. In a more preferred embodiment, the isolated serotype 24F capsular polysaccharide is sized to a weight average molecular weight of between 125 kDa and 275 kDa. In a most preferred embodiment, the isolated serotype 24F capsular polysaccharide is sized to a weight average molecular weight of between 125 kDa and 225 kDa. Preferably, the isolated serotype 24F polysaccharide is sized by mechanical homogenization, preferably by high-pressure homogenization.

[0171] In one embodiment, the isolated serotype 24F capsular polysaccharide is unsized. In one embodiment, the isolated serotype 24F capsular polysaccharide prior to conjugation has a weight average molecular weight of between 50 kDa and 500 kDa. In one embodiment, the isolated serotype 24F capsular polysaccharide prior to conjugation has a weight average molecular weight of between 75 kDa and 400 kDa. In a more preferred embodiment, the isolated serotype 24F capsular polysaccharide prior to conjugation has a weight average molecular weight of between 125 kDa and 275 kDa. In a most preferred embodiment, the isolated serotype 24F capsular polysaccharide prior to conjugation has a weight average molecular weight of between 125 kDa and 225 kDa.

[0172] The weight average molecular weight (Mw) of a saccharide prior to conjugation refers to the Mw prior to activation of the polysaccharide (i.e., after the final sizing step, but before reacting the polysaccharide with an activating agent). In the context of the present invention, the Mw of the 24F polysaccharide is not substantially altered by the activation step, and the Mw of the 24F polysaccharide incorporated into the conjugate is similar to the Mw of the various polysaccharides measured prior to activation.

[0173] In one embodiment, the serotype 24F glycoconjugate of the invention comprises a serotype 24F capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 400 kDa. In one embodiment, the weight average molecular weight (Mw) is between 120 kDa and 250 kDa. In a most preferred embodiment, the weight average molecular weight (Mw) is between 120 kDa and 200 kDa.

[0174] In some embodiments, the serotype 24F glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 10,000 kDa. In other embodiments, the serotype 24F glycoconjugates have a weight average molecular weight (Mw) between 1,500 kDa and 7,500 kDa. Preferably, the serotype 24F glycoconjugates have a weight average molecular weight (Mw) between 3,000 kDa and 6,000 kDa.

[0175] The serotype 24F glycoconjugates of the present invention can also be characterized by the saccharide to carrier protein ratio (w / w). In some embodiments, the serotype 24F polysaccharide to carrier protein ratio (w / w) in the glycoconjugate is between 0.5 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.7 and 1.5. Even more preferably, the saccharide to carrier protein ratio (w / w) is between 0.8 and 1.4.

[0176] Another method for characterizing the serotype 24F glycoconjugates of the present invention is by the number of lysine residues in the carrier protein (e.g., CRM197, DT, or TT) that become conjugated to a saccharide, which can be characterized as the extent of conjugated lysines (degree of conjugation). Evidence of lysine modification of the carrier protein by covalent attachment to a polysaccharide can be obtained by amino acid analysis using routine methods known to those skilled in the art. Conjugation results in a reduction in the number of recovered lysine residues compared to the carrier protein starting material used to generate the conjugate material. In preferred embodiments, the degree of conjugation of the serotype 24F glycoconjugates of the present invention is between 2 and 15. Preferably, the degree of conjugation of the serotype 24F glycoconjugates of the present invention is between 5 and 12.

[0177] Serotype 24F glycoconjugates and immunogenic compositions of the invention may contain free saccharides that are not covalently conjugated to a carrier protein but are nevertheless present in the glycoconjugate composition. The free saccharides may be non-covalently associated with the glycoconjugate (i.e., non-covalently bound to, adsorbed to, or entrapped in or with it).

[0178] In one embodiment, the serotype 24F glycoconjugate comprises less than about 40% free serotype 24F polysaccharide relative to the total amount of serotype 24F polysaccharide. In a preferred embodiment, the serotype 24F glycoconjugate comprises less than about 25% free serotype 24F polysaccharide relative to the total amount of serotype 24F polysaccharide.

[0179] Serotype 24F glycoconjugates can be characterized by molecular size distribution (Kd). Size-exclusion chromatography media (CL-4B) can be used to determine the relative molecular size distribution of conjugates. Size-exclusion chromatography (SEC) is used in a gravity-fed column to profile the molecular size distribution of conjugates. Large molecules excluded from pores in the media elute more rapidly than small molecules. A fraction collector is used to collect the column eluate. Fractions are tested colorimetrically by saccharide assay. To determine Kd, the column is calibrated to establish the fraction at which molecules are completely excluded (V0), (Kd = 0), and the fraction representing maximum retention (Vi), (Kd = 1). The fraction at which a particular sample attribute is reached (Ve) is related to Kd by the expression Kd = (Ve - V0) / (Vi - V0).

[0180] In one embodiment, at least 40% of the serotype 24F carbohydrates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 50% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 50% and 90% of the serotype 24F glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0181] Serotype 24F carbohydrates can also be characterized by the amount of branching ribose residues remaining in the 24F polysaccharide. As noted above, it has been found that serotype 23A polysaccharides can lose branching ribose residues (see Figure 7).

[0182] Thus, in one aspect, a Streptococcus pneumoniae serotype 24F glycoconjugate of the invention comprises a Streptococcus pneumoniae serotype 24F capsular polysaccharide, which has a ribose content of greater than 75% compared to native Streptococcus pneumoniae serotype 24F capsular polysaccharide, which is believed to have a ribose content of about 100%. In one aspect, a Streptococcus pneumoniae serotype 24F glycoconjugate of the invention comprises a Streptococcus pneumoniae serotype 24F capsular polysaccharide, which has a ribose content of greater than 90% compared to native Streptococcus pneumoniae serotype 24F capsular polysaccharide, which is believed to have a ribose content of about 100%. Preferably, the Streptococcus pneumoniae serotype 24F glycoconjugate of the invention comprises Streptococcus pneumoniae serotype 24F capsular polysaccharide, said Streptococcus pneumoniae serotype 24F capsular polysaccharide having a ribose content of greater than 95% compared to native Streptococcus pneumoniae serotype 24F capsular polysaccharide, which is believed to have a ribose content of about 100%.

[0183] In a most preferred embodiment, the Streptococcus pneumoniae serotype 24F glycoconjugate of the invention comprises Streptococcus pneumoniae serotype 24F capsular polysaccharide, said Streptococcus pneumoniae serotype 24F capsular polysaccharide having a ribose content of about 100% compared to native Streptococcus pneumoniae serotype 24F capsular polysaccharide, which is considered to have a ribose content of about 100%.

[0184] In one aspect, the serotype 24F saccharide is activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide is then coupled, either directly or via a spacer (linker) group, to a carrier protein (preferably a CRMP). 197For example, the spacer can be cystamine or cysteamine, resulting in a thiolated polysaccharide, which can be coupled to the carrier by a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyrloxy] succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g., using iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl) aminobenzoate (SlAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA), or succinimidyl 3-[bromoacetamido] propionate (SBAP)). Preferably, the cyanate ester is coupled with hexanediamine or adipic acid dihydrazide (ADH), and the amino-derivatized saccharide is attached to the carrier protein (e.g., CRM) using carbodiimide (e.g., EDAC or EDC) chemistry via carboxyl groups on the protein carrier. 197 Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348 and WO 96 / 129094.

[0185] Other suitable techniques for conjugation use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU. Many are described in International Patent Application Publication No. WO 98 / 42721. Conjugation may involve a carbonyl linker, which can be formed by reaction of the free hydroxyl group of a sugar with 1,1'-carbonyldiimidazole (CDI) (see Bethell et al. (1979) J. Biol. Chern. 254:2572-2574; Hearn et al. (1981) J. Chromatogr. 218:509-518), followed by reaction with the protein to form a carbamate bond. This may involve reducing the anomeric terminus to a primary hydroxyl group, optionally protecting / deprotecting the primary hydroxyl group, reacting the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling the CDI carbamate intermediate to an amino group on the protein.

[0186] In a preferred embodiment, the serotype 24F glycoconjugates of the invention are prepared using reductive amination chemistry. According to the invention, reductive amination involves two steps: (1) oxidizing (activating) a purified saccharide, and (2) reducing the activated saccharide and carrier protein to form the glycoconjugate.

[0187] In one aspect, the serotype 24F glycoconjugates of the invention are prepared by conjugating isolated serotype 24F capsular polysaccharide to a carrier protein by a process comprising the following steps: (a) reacting the isolated serotype 24F capsular polysaccharide with an oxidizing agent; (b) combining the activated polysaccharide of step (a) with a carrier protein; and (c) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate.

[0188] As noted above, sizing of the isolated serotype 24F capsular polysaccharide to a target molecular weight (MW) range may be performed prior to oxidation. Thus, in one aspect, the isolated serotype 24F capsular polysaccharide is sized prior to oxidation.

[0189] In a preferred embodiment, the isolated serotype 24F capsular polysaccharide is size reduced by mechanical homogenization. In one embodiment, the isolated serotype 24F polysaccharide is size reduced by high-pressure homogenization. In a most preferred embodiment, the isolated serotype 24F capsular polysaccharide is not sized by acid hydrolysis.

[0190] In one embodiment, the isolated serotype 24F capsular polysaccharide is not sized prior to oxidation. In one aspect, the oxidation step (a) is carried out at a pH between 4.5 and 6.5. Preferably, the oxidation step (a) is carried out at a pH between 5.0 and 6.0.

[0191] In one aspect, the oxidation step (a) is carried out at a pH of about 5.0. The saccharides are said to be activated after the oxidation step (a) and are called "activated polysaccharides".

[0192] In one embodiment, the activated serotype 24F polysaccharide of the present invention has a weight average molecular weight (Mw) between 50 kDa and 400 kDa. In one embodiment, the weight average molecular weight (Mw) is between 120 kDa and 250 kDa. In a most preferred embodiment, the weight average molecular weight (Mw) is between 120 kDa and 200 kDa.

[0193] In one embodiment, the activated serotype 24F polysaccharide of the present invention retains at least 75% branched ribose. In one embodiment, the activated serotype 24F polysaccharide of the present invention retains at least 90% branched ribose. In a preferred embodiment, the activated serotype 24F polysaccharide of the present invention retains at least 95% branched ribose.

[0194] In a most preferred embodiment, the activated serotype 24F polysaccharide of the present invention retains about 100% branched ribose. In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes terminal hydroxyl groups to aldehydes. In one embodiment, the oxidizing agent is periodate. For purposes of the present invention, the term "periodate" includes both periodate and periodic acid, and the term also includes metaperiodate (IO 4- ) and orthoperiodate (IO6 5- ) and various salts of periodate (e.g., sodium periodate and potassium periodate).

[0195] In a preferred embodiment, the oxidizing agent is sodium periodate. In one embodiment, the periodate used for oxidation is metaperiodate. In a most preferred embodiment, the periodate used for oxidation is sodium metaperiodate.

[0196] When a polysaccharide is reacted with periodate, the periodate oxidizes vicinal hydroxyl groups to form carbonyl or aldehyde groups, causing cleavage of C-C bonds. For this reason, the term "reacting a polysaccharide with periodate" includes the oxidation of vicinal hydroxyl groups by periodate.

[0197] In one aspect, step a) comprises reacting the polysaccharide with 0.1 to 2 molar equivalents of periodate. Preferably, step a) comprises reacting the polysaccharide with 0.5 to 1.5 molar equivalents of periodate. Most preferably, step a) comprises reacting the polysaccharide with 0.9 to 1.1 molar equivalents of periodate.

[0198] In one embodiment, the degree of oxidation of the activated serotype 24F polysaccharide (also referred to herein as the "degree of activation") is between 2 and 20. In a preferred embodiment, the degree of oxidation of the activated serotype 24F polysaccharide is between 4 and 15. In a most preferred embodiment, the degree of oxidation of the activated serotype 24F polysaccharide is 9±3.

[0199] In one aspect, the activated serotype 24F polysaccharide and carrier protein are lyophilized prior to step b). Preferably, lyophilization occurs after step a). In one aspect, the activated polysaccharide is lyophilized after step a) and the carrier protein is also lyophilized. In one aspect, the activated serotype 24F polysaccharide is lyophilized after step a) and the carrier protein is also lyophilized, and the activated polysaccharide and carrier protein are reconstituted in the same solution.

[0200] In one embodiment, the activated serotype 24F polysaccharide and the carrier protein are lyophilized independently (separate lyophilization). In one embodiment, the activated serotype 24F polysaccharide and the carrier protein are lyophilized together (co-lyophilized).

[0201] In one aspect, lyophilization occurs in the presence of a non-reducing sugar, and possible non-reducing sugars include sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, and palatinit. In one aspect, the sugar is selected from the group consisting of sucrose, trehalose, and mannitol. In one aspect, the sugar is sucrose, trehalose, or mannitol. In one aspect, the sugar is sucrose.

[0202] In one aspect, the initial weight ratio of activated serotype 24F capsular polysaccharide to carrier protein in step b) is between 2:1 and 0.5:1. In one aspect, the initial weight ratio of activated serotype 24F capsular polysaccharide to carrier protein is between 1.2:1 and 0.6:1. Preferably, the initial weight ratio of activated serotype 24F capsular polysaccharide to carrier protein is between 0.9:1 and 0.7:1.

[0203] In one embodiment, the reduction reaction (c) is carried out in an aqueous solvent. Preferably, the reduction reaction (c) is carried out in an aprotic solvent. In one embodiment, the reduction reaction (c) is carried out in the presence of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). Preferably, the reduction reaction (c) is carried out in the presence of dimethyl sulfoxide (DMSO).

[0204] In one embodiment, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethyl formamide) solvent. Most preferably, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) solvent.

[0205] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium or zinc borohydride, amine borane, such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3, or 5-ethyl-2-methylpyridine borane (PEMB), in the presence of a Bronsted acid or Lewis acid. In one embodiment, the reducing agent is sodium triacetoxyborohydride. In one embodiment, the reducing agent is sodium cyanoborohydride in the presence of nickel (see WO2018144439). In a preferred embodiment, the reducing agent is sodium cyanoborohydride.

[0206] In one embodiment, between 0.2 and 5 molar equivalents of reducing agent are used in step c). Preferably, between 0.5 and 2.5 molar equivalents of reducing agent are used in step c). Most preferably, between 1.5 and 2.5 molar equivalents of reducing agent are used in step c).

[0207] In one aspect, between about 2 molar equivalents of reducing agent are used in step c). At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugate, which can be capped using a suitable capping agent, which in one aspect is sodium borohydride (NaBH4).

[0208] In one aspect, capping is achieved by combining the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In one aspect, capping is achieved by combining the product of step c) with 1 to 3 molar equivalents of sodium borohydride.

[0209] In one aspect, capping is accomplished by combining the product of step c) with about 2 molar equivalents of sodium borohydride. After conjugation to a carrier protein, the serotype 24F glycoconjugate can be purified (concentrated with respect to the amount of saccharide-protein conjugate) by a variety of techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration operations, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. Thus, in one aspect, the process for producing a serotype 24F glycoconjugate of the invention includes a step of purifying the glycoconjugate after it has been produced. 1.5 Streptococcus pneumoniae serotype 35B glycoconjugates of the present invention In one aspect, the present invention relates to Streptococcus pneumoniae serotype 23B glycoconjugates.

[0210] The structure of the Streptococcus pneumoniae serotype 35B polysaccharide is known in the art (see, e.g., Geno K et al. (2015) Clin Microbiol Rev 28:3, 871-899).

[0211] In one aspect, the encapsulated S. pneumoniae serotype 35B saccharide used in the invention is a synthetic carbohydrate. However, in a preferred embodiment, the source of bacterial polysaccharides according to the present invention may be Streptococcus pneumoniae serotype 35B bacterial cells. Bacterial strains that can be used as a source of Streptococcus pneumoniae serotype 35B polysaccharides can be obtained from established culture collections (such as, for example, from the Streptococcus Reference Laboratory (US Centers for Disease Control and Prevention, Atlanta, GA, USA)) or from clinical specimens.

[0212] Serotype 35B saccharides can be obtained directly from bacteria using isolation procedures known to those skilled in the art, or they can be purchased commercially (e.g., from the American Type Culture Collection (ATCC), Manassas, Virginia, USA) (e.g., Reference No. ATCC 539-X, No. ATCC 540-X, No. ATCC 541-X)).

[0213] When serotype 35B saccharides are obtained directly from bacteria, the bacterial cells can be grown in a medium, preferably a soy-based medium. After fermentation of the bacterial cells that produce Streptococcus pneumoniae serotype 35B capsular polysaccharide, the bacterial cells can be lysed to produce a cell lysate. The serotype 35B polysaccharide can then be isolated from the cell lysate using purification techniques known in the art, including centrifugation, depth filtration, precipitation, ultrafiltration, treatment with activated carbon, diafiltration, and / or column chromatography (see, e.g., US2006 / 0228380, US2006 / 0228381, WO2008 / 118752, and WO2020170190). The purified serotype 35B capsular polysaccharide can be used to prepare glycoconjugates.

[0214] The isolated serotype 35B capsular saccharides obtained by purification of serotype 35B polysaccharide from S. pneumoniae lysate and, optionally, sizing of the purified polysaccharide, can be characterized by various parameters including, for example, weight average molecular weight (Mw).

[0215] The molecular weight of polysaccharides can be measured by size exclusion chromatography (SEC) coupled with a multi-angle laser light scattering detector (MALLS). In a preferred embodiment, the isolated serotype 35B capsular polysaccharide (i.e., purified prior to further processing) has a weight average molecular weight of between 100 kDa and 5000 kDa. In one embodiment, the isolated serotype 35B capsular polysaccharide has a weight average molecular weight of between 300 kDa and 2000 kDa. In a preferred embodiment, the isolated serotype 35B capsular polysaccharide has a weight average molecular weight of between 500 kDa and 1000 kDa.

[0216] Any integer within any of the above ranges is contemplated as an aspect of the present disclosure. The size of the purified serotype 35B polysaccharide can be reduced while preserving important features of the polysaccharide's structure. Mechanical or chemical sizing can be used.

[0217] However, Streptococcus pneumoniae serotype 35B polysaccharide has been found to be cleaved upon activation with periodate, a classical oxidizing agent used in commonly used reductive amination processes. Periodate oxidation occurs in the backbone of the serotype 35B polysaccharide, likely cleaving mannitol or ribitol, leading to size reduction. Activation with periodate results in a decrease in polysaccharide Mw. Therefore, when activation with periodate is used, serotype 35B capsular polysaccharide is not sized.

[0218] Thus, in one aspect, the isolated serotype 35B capsular polysaccharide is unsized. In one embodiment, the isolated serotype 35B capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 5,000 kDa. In one embodiment, the isolated serotype 35B capsular polysaccharide prior to conjugation has a weight average molecular weight of between 300 kDa and 2000 kDa. In a more preferred embodiment, the isolated serotype 35B capsular polysaccharide prior to conjugation has a weight average molecular weight of between 500 kDa and 1000 kDa.

[0219] The weight average molecular weight (Mw) of a saccharide prior to conjugation refers to the Mw prior to activation of the polysaccharide (ie, prior to reacting the polysaccharide with an activating agent). In one embodiment, the serotype 35B glycoconjugate of the invention comprises a serotype 35B capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide is between 15 kDa and 100 kDa. In one embodiment, the weight average molecular weight (Mw) is between 25 kDa and 50 kDa. In a most preferred embodiment, the weight average molecular weight (Mw) is between about 30 kDa and about 40 kDa.

[0220] In some embodiments, the serotype 35B glycoconjugates of the present invention have a weight average molecular weight (Mw) between 250 kDa and 7,500 kDa. In other embodiments, the serotype 35B glycoconjugates have a weight average molecular weight (Mw) between 500 kDa and 5,000 kDa. Preferably, the serotype 35B glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 4,000 kDa.

[0221] The serotype 35B glycoconjugates of the present invention can be characterized by their saccharide to carrier protein ratio (w / w). In some embodiments, the serotype 35B polysaccharide to carrier protein ratio (w / w) in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.4 and 2.0. Even more preferably, the saccharide to carrier protein ratio (w / w) is between 0.5 and 1.5.

[0222] Another method for characterizing the serotype 35A glycoconjugates of the invention is by the number of lysine residues in the carrier protein (e.g., CRM197, DT, or TT) that become conjugated to a saccharide, which can be characterized as the extent of conjugated lysines (degree of conjugation). Evidence of lysine modification of the carrier protein by covalent attachment to a polysaccharide can be obtained by amino acid analysis using routine methods known to those of skill in the art. Conjugation results in a reduction in the number of recovered lysine residues compared to the carrier protein starting material used to generate the conjugate material. In preferred embodiments, the degree of conjugation of the serotype 35B glycoconjugates of the invention is between 2 and 15. Preferably, the degree of conjugation of the serotype 35B glycoconjugates of the invention is between 5 and 10.

[0223] Serotype 35B glycoconjugates and immunogenic compositions of the invention may contain free saccharides that are not covalently conjugated to a carrier protein but are nevertheless present in the glycoconjugate composition. The free saccharides may be non-covalently associated with (i.e., non-covalently bound to, adsorbed to, or entrapped in or with) the glycoconjugate.

[0224] In one embodiment, the serotype 35B glycoconjugate comprises less than about 40% free serotype 35B polysaccharide relative to the total amount of serotype 35B polysaccharide. In one embodiment, the serotype 35B glycoconjugate comprises less than about 20% free serotype 35B polysaccharide relative to the total amount of serotype 35B polysaccharide. In a preferred embodiment, the serotype 35B glycoconjugate comprises less than about 10% free serotype 35B polysaccharide relative to the total amount of serotype 15A polysaccharide.

[0225] Serotype 35B glycoconjugates can be characterized by molecular size distribution (Kd). Size-exclusion chromatography media (CL-4B) can be used to determine the relative molecular size distribution of conjugates. Size-exclusion chromatography (SEC) is used in a gravity-fed column to profile the molecular size distribution of conjugates. Large molecules excluded from pores in the media elute more rapidly than small molecules. A fraction collector is used to collect the column eluate. Fractions are tested colorimetrically by saccharide assay. To determine Kd, the column is calibrated to establish the fraction at which molecules are completely excluded (V0), (Kd = 0), and the fraction representing maximum retention (Vi), (Kd = 1). The fraction at which a particular sample attribute is reached (Ve) is related to Kd by the expression Kd = (Ve - V0) / (Vi - V0).

[0226] In one embodiment, at least 40% of the serotype 35B glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 60% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 50% and 80% of the serotype 35B glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0227] In one aspect, serotype 35B saccharides are activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form cyanate esters. The activated polysaccharides are then coupled, either directly or via a spacer (linker) group, to a carrier protein (preferably a CRMP). 197 For example, the spacer can be cystamine or cysteamine, resulting in a thiolated polysaccharide, which can be coupled to the carrier by a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyrloxy] succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g., using iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl) aminobenzoate (SlAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA), or succinimidyl 3-[bromoacetamido] propionate (SBAP)). Preferably, the cyanate ester is coupled with hexanediamine or adipic acid dihydrazide (ADH), and the amino-derivatized saccharide is attached to the carrier protein (e.g., CRM) using carbodiimide (e.g., EDAC or EDC) chemistry via carboxyl groups on the protein carrier. 197 Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348 and WO 96 / 129094.

[0228] Other suitable techniques for conjugation use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU. Many are described in International Patent Application Publication No. WO 98 / 42721. Conjugation may involve a carbonyl linker, which can be formed by reaction of the free hydroxyl group of a sugar with 1,1'-carbonyldiimidazole (CDI) (see Bethell et al. (1979) J. Biol. Chern. 254:2572-2574; Hearn et al. (1981) J. Chromatogr. 218:509-518), followed by reaction with the protein to form a carbamate bond. This may involve reducing the anomeric terminus to a primary hydroxyl group, optionally protecting / deprotecting the primary hydroxyl group, reacting the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling the CDI carbamate intermediate to an amino group on the protein.

[0229] In a preferred embodiment, the serotype 35B glycoconjugates of the present invention are prepared using reductive amination chemistry. According to the present invention, reductive amination involves two steps: (1) oxidizing (activating) a purified saccharide; and (2) reducing the activated saccharide and carrier protein to form the glycoconjugate. In one aspect, the serotype 35B glycoconjugate of the invention is prepared by conjugating an isolated serotype 35B capsular polysaccharide to a carrier protein by a process comprising the steps of: (a) reacting the isolated serotype 35B capsular polysaccharide with an oxidizing agent; (b) combining the activated polysaccharide of step (a) with a carrier protein; and (c) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form the glycoconjugate.

[0230] Preferably, the isolated serotype 35B capsular polysaccharide is not sized prior to oxidation. In a preferred embodiment, step (a) is quenched by adding a quenching agent to stop the oxidation.

[0231] Thus, in a preferred embodiment, the serotype 35B glycoconjugate of the present invention is prepared by conjugating isolated serotype 35B capsular polysaccharide to a carrier protein by a process comprising the following steps: (a) reacting said isolated serotype 35B capsular polysaccharide with an oxidizing agent; (a') quenching the oxidation reaction by adding a quenching agent, resulting in activated serotype 35B capsular polysaccharide; (b) combining the activated polysaccharide of step (a') with a carrier protein; and (c) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form the glycoconjugate.

[0232] In one aspect, the oxidation step (a) is carried out at a pH between 5.0 and 7.0. Preferably, the oxidation step (a) is carried out at a pH between 5.5 and 6.5. In one aspect, the oxidation step (a) is carried out at a pH of 6.0.

[0233] After the oxidation steps (a)-(a'), the saccharides are said to be activated and are called "activated polysaccharides". In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes terminal hydroxyl groups to aldehydes. In one embodiment, the oxidizing agent is periodate. For purposes of the present invention, the term "periodate" includes both periodate and periodic acid, and the term also includes metaperiodate (IO 4- ) and orthoperiodate (IO6 5- ) and various salts of periodate (e.g., sodium periodate and potassium periodate).

[0234] In a preferred embodiment, the oxidizing agent is sodium periodate. In one embodiment, the periodate used for oxidation is metaperiodate. In a most preferred embodiment, the periodate used for oxidation is sodium metaperiodate.

[0235] When a polysaccharide is reacted with periodate, the periodate oxidizes vicinal hydroxyl groups to form carbonyl or aldehyde groups, causing cleavage of C-C bonds. For this reason, the term "reacting a polysaccharide with periodate" includes the oxidation of vicinal hydroxyl groups by periodate.

[0236] In one aspect, step a) comprises reacting the polysaccharide with 0.05 to 0.2 molar equivalents of periodate. Preferably, step a) comprises reacting the polysaccharide with 0.09 to 0.11 molar equivalents of periodate. Most preferably, step a) comprises reacting the polysaccharide with about 0.1 molar equivalents of periodate.

[0237] In one aspect, the quenching agent is selected from a vicinal diol, a 1,2-amino alcohol, an amino acid, glutathione, a sulfite, a bisulfite, a dithionite, a metabisulfite, a thiosulfate, a phosphite, a hypophosphite, or a phosphorous acid.

[0238] In one embodiment, the quenching agent has the formula (I):

[0239] [ka]

[0240] [In the formula, R 1 is selected from H, methyl, ethyl, propyl or isopropyl. It is a 1,2-amino alcohol.

[0241] In one aspect, the quenching agent is selected from sodium and potassium salts of sulfite, bisulfite, dithionite, metabisulfite, thiosulfate, phosphorous acid, hypophosphorous acid, or phosphorous acid.

[0242] In one aspect, the quenching agent is an amino acid. In such an aspect, the amino acid may be selected from serine, threonine, cysteine, cystine, methionine, proline, hydroxyproline, tryptophan, tyrosine, and histidine.

[0243] In one aspect, the quenching agent is a sulfite, such as bisulfite, dithionite, metabisulfite, or thiosulfate. In one embodiment, the quenching agent is a compound containing two vicinal hydroxyl groups (vicinal diol), ie, two hydroxyl groups covalently linked to two adjacent carbon atoms.

[0244] Preferably, the quenching agent has the formula (II):

[0245] [ka]

[0246] [In the formula, R 1 and R 2 are each independently selected from H, methyl, ethyl, propyl, or isopropyl. is a compound of

[0247] In a preferred embodiment, the quenching agent is glycerol, ethylene glycol, propane-1,2-diol, butane-1,2-diol or butane-2,3-diol or ascorbic acid. In a most preferred embodiment, the quenching agent is butane-2,3-diol.

[0248] In a preferred embodiment, the isolated serotype 35B polysaccharide is activated by a process comprising the following steps: (a) reacting isolated serotype 35B polysaccharide with periodate; (b) quenching the oxidation reaction by adding butane-2,3-diol, resulting in activated serotype 35B polysaccharide.

[0249] After the step of oxidation of the polysaccharide, the polysaccharide is said to be activated and is hereinafter referred to as "activated polysaccharide". In one embodiment, the activated serotype 35B polysaccharide of the present invention has a weight average molecular weight (Mw) between 15 kDa and 100 kDa. In one embodiment, the weight average molecular weight (Mw) is between 25 kDa and 50 kDa. In a most preferred embodiment, the weight average molecular weight (Mw) is between 30 kDa and 40 kDa.

[0250] In one embodiment, the degree of oxidation of the activated serotype 35B polysaccharide (also referred to herein as the "degree of activation") is between 2 and 20. In a preferred embodiment, the degree of oxidation of the activated serotype 35B polysaccharide is between 4 and 15. In a most preferred embodiment, the degree of oxidation of the activated serotype 35B polysaccharide is 9±3.

[0251] In one aspect, the activated serotype 35B polysaccharide and carrier protein are lyophilized prior to step b). Preferably, lyophilization occurs after step a). In one aspect, the activated polysaccharide is lyophilized after step a) and the carrier protein is also lyophilized. In one aspect, the activated serotype 35B polysaccharide is lyophilized after step a) and the carrier protein is also lyophilized, and the activated polysaccharide and carrier protein are reconstituted in the same solution.

[0252] In one embodiment, the activated serotype 35B polysaccharide and the carrier protein are lyophilized independently (separate lyophilization). In one embodiment, the activated serotype 35B polysaccharide and the carrier protein are lyophilized together (co-lyophilized).

[0253] In one aspect, lyophilization occurs in the presence of a non-reducing sugar, and possible non-reducing sugars include sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, and palatinit. In one aspect, the sugar is selected from the group consisting of sucrose, trehalose, and mannitol. In one aspect, the sugar is sucrose, trehalose, or mannitol. In one aspect, the sugar is sucrose.

[0254] In one aspect, the initial weight ratio of activated serotype 35B capsular polysaccharide to carrier protein in step b) is between 2:1 and 0.5:1. In one aspect, the initial weight ratio of activated serotype 35B capsular polysaccharide to carrier protein is between 1.2:1 and 0.6:1. Preferably, the initial weight ratio of activated serotype 35B capsular polysaccharide to carrier protein is between 0.9:1 and 0.7:1.

[0255] In one embodiment, the reduction reaction (c) is carried out in an aqueous solvent. Preferably, the reduction reaction (c) is carried out in an aprotic solvent. In one embodiment, the reduction reaction (c) is carried out in the presence of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). Preferably, the reduction reaction (c) is carried out in the presence of dimethyl sulfoxide (DMSO).

[0256] In one embodiment, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethyl formamide) solvent. Most preferably, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) solvent.

[0257] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium or zinc borohydride, amine borane, such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3, or 5-ethyl-2-methylpyridine borane (PEMB), in the presence of a Bronsted acid or Lewis acid. In one embodiment, the reducing agent is sodium triacetoxyborohydride. In one embodiment, the reducing agent is sodium cyanoborohydride in the presence of nickel (see WO2018144439). In a preferred embodiment, the reducing agent is sodium cyanoborohydride.

[0258] In one embodiment, between 0.2 and 5 molar equivalents of reducing agent are used in step c). Preferably, between 0.5 and 1.5 molar equivalents of reducing agent are used in step c). Most preferably, between 0.9 and 1.1 molar equivalents of reducing agent are used in step c).

[0259] At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugate, which can be capped using a suitable capping agent, which in one aspect is sodium borohydride (NaBH4).

[0260] In one aspect, capping is achieved by combining the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In one aspect, capping is achieved by combining the product of step c) with 1 to 3 molar equivalents of sodium borohydride.

[0261] After conjugation to a carrier protein, the serotype 35B glycoconjugates can be purified (concentrated with respect to the amount of saccharide-protein conjugate) by a variety of techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration operations, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. Thus, in one aspect, the process for producing the serotype 35B glycoconjugates of the invention includes a purification step after production. 1.6 Streptococcus pneumoniae serotype 3 glycoconjugates of the present invention In one aspect, the invention relates to a composition comprising a Streptococcus pneumoniae serotype 3 glycoconjugate.

[0262] The structure of the Streptococcus pneumoniae serotype 3 polysaccharide is known in the art. The serotype 3 polysaccharide repeating unit consists of a linear disaccharide unit with one glycopyranose (Glcp) and one glucuronic acid (GlcpA) (see, e.g., Geno K et al. (2015) Clin Microbiol Rev 28:3, 871-899).

[0263] In one aspect, the capsular Streptococcus pneumoniae serotype 3 saccharide used in the present invention is a synthetic carbohydrate. Preparation of synthetic Streptococcus pneumoniae type 3 capsular saccharide may be carried out, for example, as disclosed in WO2017178664 or WO2015040140.

[0264] However, in a preferred embodiment, the source of bacterial polysaccharides according to the present invention may be bacterial cells of Streptococcus pneumoniae serotype 3. Bacterial strains that can be used as a source of Streptococcus pneumoniae serotype 3 polysaccharides can be obtained from established culture collections (such as, for example, from the Streptococcus Reference Laboratory (US Centers for Disease Control and Prevention, Atlanta, GA, USA)) or from clinical specimens.

[0265] Serotype 3 polysaccharides can be obtained directly from bacteria using isolation procedures known to those of skill in the art (e.g., methods disclosed in US2006 / 0228380, US2006 / 0228381, US2007 / 0184071, US2007 / 0184072, US2007 / 0231340, and US2008 / 0102498 and WO2008 / 118752). They can also be produced using synthetic protocols known to those of skill in the art. They can also be purchased commercially (e.g., from the American Type Culture Collection (ATCC), Manassas, Virginia, USA) (e.g., reference numbers ATCC 172-X or ATCC 33-X).

[0266] When serotype 3 polysaccharide is obtained directly from bacteria, the bacterial cells can be grown in a medium, preferably a soy-based medium. After fermentation of the bacterial cells that produce Streptococcus pneumoniae serotype 3 capsular polysaccharide, the bacterial cells can be lysed to produce a cell lysate. The serotype 3 polysaccharide can then be isolated from the cell lysate using purification techniques known in the art, including centrifugation, depth filtration, precipitation, ultrafiltration, treatment with activated carbon, diafiltration, and / or column chromatography (see, for example, US2006 / 0228380, US2006 / 0228381, and WO2008 / 118752). The purified serotype 3 capsular polysaccharide can then be used to prepare immunogenic conjugates.

[0267] The isolated serotype 3 capsular polysaccharide obtained by purification of serotype 3 polysaccharide from S. pneumoniae lysate and, optionally, sizing of the purified polysaccharide can be characterized by various parameters including, for example, weight average molecular weight (Mw).

[0268] The molecular weight of polysaccharides can be measured by size exclusion chromatography (SEC) coupled with a multi-angle laser light scattering detector (MALLS). In one embodiment, the isolated serotype 3 capsular polysaccharide (i.e., purified prior to further processing) has a weight average molecular weight of between 5 kDa and 5,000 kDa. In one embodiment, the isolated capsular polysaccharide has a weight average molecular weight of between 100 kDa and 4,000 kDa. In a preferred embodiment, the isolated capsular polysaccharide has a weight average molecular weight of between 1,00 kDa and 3,500 kDa.

[0269] Preferably, sizing of the polysaccharide to a target molecular weight range is performed prior to conjugation to the carrier protein to produce a serotype 3 conjugate with advantageous filterability characteristics, immunogenicity, and / or yield. Advantageously, the size of the purified serotype 3 polysaccharide is reduced while preserving important structural features of the polysaccharide. Mechanical or chemical sizing can be used.

[0270] In one embodiment, the size of the purified serotype 3 polysaccharide is reduced by chemical hydrolysis. Chemical hydrolysis may be carried out using a weak acid (e.g., acetic acid, formic acid, propanoic acid). In one embodiment, chemical hydrolysis is carried out using formic acid. In one embodiment, chemical hydrolysis is carried out using propanoic acid. In a preferred embodiment, chemical hydrolysis is carried out using acetic acid. Chemical hydrolysis may also be carried out using a dilute strong acid (e.g., dilute hydrochloric acid, dilute sulfuric acid, dilute phosphoric acid, dilute nitric acid, or dilute perchloric acid). In one embodiment, chemical hydrolysis is carried out using dilute hydrochloric acid. In one embodiment, chemical hydrolysis is carried out using dilute sulfuric acid. In one embodiment, chemical hydrolysis is carried out using dilute phosphoric acid. In one embodiment, chemical hydrolysis is carried out using dilute nitric acid. In one embodiment, chemical hydrolysis is carried out using dilute perchloric acid.

[0271] The size of purified serotype 3 polysaccharide can also be reduced by mechanical homogenization. In one embodiment, the size of purified serotype 3 polysaccharide is reduced by high-pressure homogenization. High-pressure homogenization achieves high shear rates by pumping the process stream through channels of sufficiently small dimensions. The shear rate can be increased by using greater applied homogenization pressure, and the exposure time can be increased by recirculating the feed stream through the homogenizer. The high-pressure homogenization process can be suitable for reducing the size of purified serotype 3 polysaccharide while preserving the structural characteristics of the polysaccharide.

[0272] In one aspect, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 5 kDa and 1000 kDa. In one aspect, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 50 kDa and 300 kDa. In a preferred aspect, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 100 kDa and 300 kDa.

[0273] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between about 200 kDa and about 300 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between about 100 kDa and about 200 kDa.

[0274] In one aspect, the isolated serotype 3 capsular polysaccharide is unsized. In one aspect, the serotype 3 glycoconjugate of the invention comprises a serotype 3 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. Preferably, the weight average molecular weight (Mw) is between 100 kDa and 300 kDa.

[0275] The weight average molecular weight (Mw) of a serotype 3 polysaccharide before conjugation refers to the Mw before activation of the serotype 3 polysaccharide (i.e., after the final sizing step, but before the polysaccharide is reacted with an activating agent). In the context of the present invention, the Mw of the serotype 3 polysaccharide is not substantially altered by the activation step, and the Mw of the serotype 3 polysaccharide incorporated into the conjugate is similar to the Mw of the various polysaccharides measured before activation.

[0276] In one aspect, the serotype 3 glycoconjugate of the invention comprises a serotype 3 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 100 kDa and 200 kDa.

[0277] In one aspect, the serotype 3 glycoconjugate of the invention comprises a serotype 3 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 200 kDa and 300 kDa.

[0278] In some embodiments, the serotype 3 glycoconjugates of the present invention have a weight average molecular weight (Mw) between 250 kDa and 20,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) between 500 kDa and 10,000 kDa. Preferably, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) between 500 kDa and 5,000 kDa.

[0279] In one embodiment, the serotype 3 glycoconjugate has a weight average molecular weight (Mw) of between 600 kDa and 3,000 kDa. The molecular weight of polysaccharides can be measured by size exclusion chromatography (SEC) coupled with a multi-angle laser light scattering detector (MALLS).

[0280] Another method for characterizing the serotype 3 glycoconjugates of the invention involves characterizing the carrier protein (e.g., CRMP) that becomes conjugated to the saccharide. 197 The degree of conjugation is determined by the number of lysine residues in the carrier protein (or SCP), which can be characterized as the extent of conjugated lysines (degree of conjugation). Evidence of lysine modification of the carrier protein by covalent attachment to the polysaccharide can be obtained by amino acid analysis using routine methods known to those skilled in the art. Conjugation results in a reduction in the number of recovered lysine residues compared to the carrier protein starting material used to generate the conjugated material. In preferred embodiments, the degree of conjugation of the serotype 3 glycoconjugates of the invention is between 2 and 15.

[0281] In preferred embodiments, the degree of conjugation of the serotype 3 glycoconjugates of the invention is between 4 and 7. In some such embodiments, the carrier protein is a CRM 197 In other such embodiments, the carrier protein is SCP.

[0282] The serotype 3 glycoconjugates of the invention can be characterized by the ratio of saccharide to carrier protein (w / w). In some embodiments, the ratio of serotype 3 polysaccharide to carrier protein (w / w) in the glycoconjugate is between 0.5 and 3.0. In other embodiments, the ratio of saccharide to carrier protein (w / w) is between 0.5 and 1.5. In preferred embodiments, the ratio of serotype 3 capsular polysaccharide to carrier protein in the conjugate is between 0.9 and 1.1.

[0283] Serotype 3 glycoconjugates of the invention can also be characterized by the number of covalent linkages between the carrier protein and the polysaccharide as a function of saccharide repeat unit. In one embodiment, a serotype 3 glycoconjugate of the invention comprises at least one covalent linkage between the carrier protein and the polysaccharide for every tetrasaccharide repeat unit of the polysaccharide. In another embodiment, a covalent linkage between the carrier protein and the polysaccharide occurs at least once for every 10 polysaccharide saccharide repeat units. In another embodiment, a covalent linkage between the carrier protein and the polysaccharide occurs at least once for every 15 polysaccharide saccharide repeat units. In a further embodiment, a covalent linkage between the carrier protein and the polysaccharide occurs at least once for every 25 polysaccharide saccharide repeat units. In a further embodiment, a covalent linkage between the carrier protein and the polysaccharide occurs at least once for every 50 polysaccharide saccharide repeat units. In yet a further embodiment, a covalent linkage between the carrier protein and the polysaccharide occurs at least once for every 100 polysaccharide saccharide repeat units.

[0284] In other embodiments, the serotype 3 glycoconjugates of the invention comprise at least one covalent linkage between the carrier protein and the polysaccharide for every 5-10 saccharide repeat units of the polysaccharide. In other embodiments, the serotype 3 glycoconjugates of the invention comprise at least one covalent linkage between the carrier protein and the polysaccharide for every 10-20 saccharide repeat units of the polysaccharide.

[0285] In some embodiments, the carrier protein is a CRM 197 and CRM 197The covalent linkage between the SCP and the polysaccharide occurs at least once for every 4, 10, 15, or 25 saccharide repeating units of the polysaccharide. In frequent embodiments, the carrier protein is an SCP, and the covalent linkage between the SCP and the polysaccharide occurs at least once for every 4, 10, 15, or 25 saccharide repeating units of the polysaccharide.

[0286] The serotype 3 glycoconjugates and immunogenic compositions of the invention may contain free saccharides that are not covalently conjugated to a carrier protein but are nevertheless present in the glycoconjugate composition. The free saccharides may be non-covalently associated with the glycoconjugate (i.e., non-covalently bound to, adsorbed to, or entrapped in or with it).

[0287] In preferred embodiments, the serotype 3 glycoconjugates comprise less than about 50% free serotype 3 polysaccharide relative to the total amount of serotype 3 polysaccharide. In preferred embodiments, the serotype 3 glycoconjugates comprise less than about 40% free serotype 3 polysaccharide relative to the total amount of serotype 3 polysaccharide. In even more preferred embodiments, the serotype 3 glycoconjugates comprise less than about 25% free serotype 3 polysaccharide relative to the total amount of serotype 3 polysaccharide. In even more preferred embodiments, the serotype 3 glycoconjugates comprise less than about 20% free serotype 3 polysaccharide relative to the total amount of serotype 3 polysaccharide. In even more preferred embodiments, the serotype 3 glycoconjugates comprise less than about 15% free serotype 3 polysaccharide relative to the total amount of serotype 3 polysaccharide.

[0288] Serotype 3 glycoconjugates have a molecular size distribution (K d ) can be characterized. Size exclusion chromatography media (CL-4B) can be used to determine the relative molecular size distribution of the conjugates. Size exclusion chromatography (SEC) is used in a gravity-fed column to profile the molecular size distribution of the conjugates. Large molecules excluded from the pores in the media elute more quickly than small molecules. A fraction collector is used to collect the column eluate. Fractions are tested colorimetrically by sugar assay. Kd To determine the fraction at which a molecule is completely excluded (V0), the column is calibrated (K d = 0), and the fraction representing maximum retention (V i ), (K d = 1). The fraction at which a particular sample characteristic is reached (V e ) is K d =(V e -V0) / (V i -V0) is related to Kd in terms of

[0289] In a preferred embodiment, at least 30% of the serotype 3 glycoconjugates have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, at least 40% of the glycoconjugates have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of the serotype 3 glycoconjugates have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, at least 60% of the serotype 3 glycoconjugates have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, between 50% and 80% of the serotype 3 glycoconjugates have a K of 0.3 or less in the CL-4B column. d In a preferred embodiment, between 65% and 80% of the serotype 3 glycoconjugates have a K of 0.3 or less in the CL-4B column. d It has.

[0290] In one aspect, the serotype 3 glycoconjugates of the invention are prepared using reductive amination chemistry (see WO2006110381, WO2008143709, PCT / IB2022 / 054920).

[0291] According to the present invention, reductive amination involves two steps: (1) oxidizing (activating) purified saccharides; and (2) oxidizing the activated saccharides and carrier proteins (e.g., CRMs). 197, TT, or SCP) to form a glycoconjugate.

[0292] As noted above, sizing of the polysaccharide to a target molecular weight (MW) range may be performed prior to oxidation. Thus, in one aspect, the isolated polysaccharide is sized prior to oxidation.

[0293] In one aspect, the isolated polysaccharide is sized to any of the target molecular weight (MW) ranges defined above. In one aspect, the isolated serotype 3 capsular polysaccharide is conjugated to a carrier protein by a process comprising the following steps: (a) reacting the isolated polysaccharide with an oxidizing agent; (b) combining the activated polysaccharide of step (a) with a carrier protein; and (c) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate.

[0294] The saccharides are said to be activated after the oxidation step (a) and are called "activated polysaccharides". In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes terminal hydroxyl groups to aldehydes. In one embodiment, the oxidizing agent is periodate. For purposes of the present invention, the term "periodate" includes both periodate and periodic acid, and the term also includes metaperiodate (IO 4- ) and orthoperiodate (IO6 5- ) and various salts of periodate (e.g., sodium periodate and potassium periodate).

[0295] In one embodiment, the oxidizing agent is periodate in the presence of a divalent cation (see WO2008 / 143709). In one embodiment, the oxidizing agent is periodic acid. In one embodiment, the oxidizing agent is periodic acid in the presence of a divalent cation. In one embodiment, the oxidizing agent is Mg 2+ In one embodiment, the oxidizing agent is periodate in the presence of Ca2+ In one embodiment, the oxidizing agent is orthoperiodate.

[0296] In one embodiment, the oxidizing agent is sodium periodate. In one embodiment, the periodate used for oxidation is metaperiodate. In one embodiment, the periodate used for oxidation is sodium metaperiodate.

[0297] When a polysaccharide is reacted with periodate, the periodate oxidizes vicinal hydroxyl groups to form carbonyl or aldehyde groups, causing cleavage of C-C bonds. For this reason, the term "reacting a polysaccharide with periodate" includes the oxidation of vicinal hydroxyl groups by periodate.

[0298] In one aspect, step a) comprises reacting the polysaccharide with 0.01 to 2 molar equivalents of periodate. Preferably, step a) comprises reacting the polysaccharide with 0.1 to 2 molar equivalents of periodate.

[0299] In one aspect, step a) comprises reacting the polysaccharide with 0.01 to 2 molar equivalents of periodic acid. Preferably, step a) comprises reacting the polysaccharide with 0.2 to 2 molar equivalents of periodic acid.

[0300] In a preferred embodiment, the degree of oxidation of the activated serotype 3 polysaccharide (also referred to herein as the "degree of activation") is between 2 and 30. In a preferred embodiment, the degree of oxidation of the activated serotype 3 polysaccharide is between 2 and 20.

[0301] In one embodiment, the degree of oxidation of the activated serotype 3 polysaccharide is between 2 and 8. In one embodiment, the degree of oxidation of the activated serotype 3 polysaccharide is between 11 and 19. In one embodiment, the activated polysaccharide and carrier protein are lyophilized prior to step b). Preferably, lyophilization occurs after step a). In one embodiment, the activated polysaccharide is lyophilized after step a) and the carrier protein is also lyophilized.

[0302] In one aspect, the activated polysaccharide is lyophilized after step a), the carrier protein is also lyophilized, and the activated polysaccharide and carrier protein are reconstituted in the same solution, which serves to combine the activated polysaccharide and carrier protein together.

[0303] In one embodiment, the activated polysaccharide and the carrier protein are lyophilized independently (separate lyophilization). In one embodiment, the activated polysaccharide and the carrier protein are lyophilized together (co-lyophilization).

[0304] In one aspect, lyophilization occurs in the presence of a non-reducing sugar, and possible non-reducing sugars include sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, and palatinit. In one aspect, the sugar is selected from the group consisting of sucrose, trehalose, and mannitol. In one aspect, the sugar is sucrose, trehalose, or mannitol. In one aspect, the sugar is trehalose. In one aspect, the sugar is sucrose.

[0305] In one aspect, the initial weight ratio of activated serotype 3 capsular polysaccharide to carrier protein in step b) is between 4:1 and 0.1:1. In one aspect, the initial weight ratio of activated serotype 3 capsular polysaccharide to carrier protein is between 2:1 and 0.4:1.

[0306] In one embodiment, the reduction reaction (c) is carried out in an aqueous solvent. In one embodiment, the reduction reaction (c) is carried out in an aprotic solvent. In one embodiment, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) solvent.

[0307] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium or zinc borohydride, an amine borane, such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe, in the presence of a Bronsted acid or a Lewis acid. i PrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridineborane (PEMB). In one embodiment, the reducing agent is sodium triacetoxyborohydride. In a preferred embodiment, the reducing agent is sodium cyanoborohydride. In one embodiment, the reducing agent is sodium cyanoborohydride in the presence of nickel (see WO2018144439).

[0308] In one embodiment, in step c), 0.2 to 20 molar equivalents of reducing agent are used. In one embodiment, in step c), 0.5 to 3 molar equivalents of reducing agent are used. At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugate, which can be capped using a suitable capping agent, which in one aspect is sodium borohydride (NaBH4).

[0309] In one aspect, capping is achieved by combining the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In one aspect, capping is achieved by combining the product of step c) with 1 to 3 molar equivalents of sodium borohydride.

[0310] In one embodiment, the glycoconjugates of the invention are prepared using CDI and / or CDT chemistry (see PCT / IB2022 / 054920). CDI and / or CDT chemistry involves two steps: (1) reacting isolated saccharides with CDI and / or CDT in an aprotic solvent to produce activated saccharides (activation); and (2) coupling the activated saccharides to a carrier protein (e.g., CRM). 197or SCP) to form a glycoconjugate.

[0311] In one aspect, the activating agent in step (1) is 1,1'-carbonyldiimidazole (CDI). In one aspect, the activating agent in step (1) is 1,1'-carbonyl-di-(1,2,4-triazole) (CDT).

[0312] In one aspect, the isolated serotype 3 capsular polysaccharide is conjugated to a carrier protein by a process comprising the following steps: (a) reacting the isolated polysaccharide with CDI and / or CDT in an aprotic solvent; (b) reacting the activated polysaccharide of step (a) with a carrier protein in an aprotic solvent to form a glycoconjugate.

[0313] In one embodiment, the serotype 3 glycoconjugates of the invention are prepared using click chemistry (see, e.g., PCT / IB2022 / 054914). According to the present invention, click chemistry involves three steps: (a) reacting isolated serotype 3 capsular polysaccharide with a carbonic acid derivative and an azide linker in an aprotic solvent to produce an activated azidopolysaccharide (polysaccharide activation); (b) reacting a carrier protein with a drug bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group, whereby the NHS moiety reacts with the amino group to form an amide bond, thereby yielding an alkyne-functionalized carrier protein (carrier protein activation); and (c) Cu +1 reacting the activated azidopolysaccharide of step (a) with the activated alkyne-carrier protein of step (b) via a mediated azide-alkyne cycloaddition reaction to form a glycoconjugate.

[0314] After step (a), the polysaccharide is said to be activated and is referred to herein as an "activated polysaccharide" or an "activated azidopolysaccharide." After step (b), the support is said to be activated and is called an "activated support."

[0315] As mentioned above, prior to activation (a), sizing of the polysaccharide to a target molecular weight (MW) range may be carried out. Thus, in one aspect, the isolated polysaccharide is sized prior to activation with a carbonate derivative and an azide linker.

[0316] In one aspect, the isolated polysaccharide is sized to any of the target molecular weight (MW) ranges defined above. In one embodiment, the carbonic acid derivative is 1,1'-carbonyldiimidazole (CDI) or 1,1'-carbonyl-di-(1,2,4-triazole) (CDT). Preferably, the carbonic acid derivative is 1,1'-carbonyldiimidazole (CDI).

[0317] In one embodiment, the azide linker has the formula (I): H2N-X-N3(I) [Wherein X is CH2(CH2) n , (CH2CH2O) m CH2CH2, NHCO(CH2) n , NHCO(CH2CH2O) m CH2CH2, OCH2(CH2) n and O(CH2CH2O) m CH2CH2, n is selected from 1 to 10, and m is selected from 1 to 4. is a compound of

[0318] In one embodiment, the azido linker has the formula (II):

[0319] [ka]

[0320] is a compound of In one embodiment, the azido linker is 3-azido-propylamine. In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is an agent having an N-hydroxysuccinimide (NHS) moiety and a terminal alkyne.

[0321] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is an agent having an N-hydroxysuccinimide (NHS) moiety and a cycloalkyne.

[0322] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group has the formula (III):

[0323] [ka]

[0324] [Wherein X is CH2O(CH2) n CH2C=O and CHO(CH2CH2O) m (CH2) n CH2C=O, n is selected from 0 to 10, and m is selected from 0 to 4. is a compound of

[0325] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group has formula (IV):

[0326] [ka]

[0327] is a compound of In one aspect, step a) comprises reacting a polysaccharide with a carbonic acid derivative, followed by reacting the carbonic acid-activated polysaccharide with an azide linker in an aprotic solvent to produce an activated azido polysaccharide.

[0328] In one embodiment, in step a) the isolated polysaccharide is reacted with a carbonic acid derivative in an aprotic solvent. In a preferred embodiment, the isolated polysaccharide is reacted with a carbonic acid derivative in a solution consisting essentially of dimethyl sulfoxide (DMSO).

[0329] In a preferred embodiment, the isolated polysaccharide is reacted with CDI in dimethyl sulfoxide (DMSO). In one embodiment, the isolated polysaccharide is reacted with CDI in anhydrous DMSO. Once the polysaccharide is reacted with the carbonate derivative, the carbonate derivative is finally quenched with water, and then the polysaccharide activated by the carbonate derivative is reacted with an azide linker.

[0330] In one aspect, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an azide linker in an amount between 0.01 and 10 molar equivalents relative to the amount of polysaccharide repeating units (molar equivalents of RU) of the activated polysaccharide.

[0331] In one embodiment, the conjugation reaction c) is carried out in an aqueous buffer solution. In one embodiment, the conjugation reaction c) is carried out in an aqueous buffer solution in the presence of copper(I) as a catalyst. In one embodiment, the conjugation reaction c) is carried out in an aqueous buffer solution in the presence of an oxidizing agent and copper(I) as a catalyst. In a preferred embodiment, the conjugation reaction c) is carried out in an aqueous buffer solution in the presence of copper(I) as a catalyst and ascorbate as an oxidizing agent. In one embodiment, THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) and aminoguanidine may be further added to protect the protein from side reactions. Thus, in a preferred embodiment, the conjugation reaction c) is carried out in an aqueous buffer solution in the presence of copper(I) as a catalyst and ascorbate as an oxidizing agent, and the reaction mixture further comprises THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) and aminoguanidine.

[0332] After the click conjugation reaction, unreacted azide groups may remain in the conjugate, which may be capped using a suitable azide group capping agent. Thus, in one embodiment, after step c), unreacted azide groups in the conjugate are capped using a suitable azide group capping agent. In one embodiment, the azide group capping agent is a drug having an alkyne group. In one embodiment, the azide group capping agent is a drug having a terminal alkyne. In one embodiment, the azide group capping agent is a drug having a cycloalkyne.

[0333] In one embodiment, the azide group capping agent has the formula (V): ≡-N-OH (V) [Wherein X is (CH2) n and n is selected from 1 to 15. is a compound of

[0334] In one embodiment, the azide group capping agent is propargyl alcohol. Thus, in one aspect, after step (c), the process further comprises capping any remaining unreacted azide groups in the conjugate with an azide group capping agent.

[0335] After the click conjugation reaction, unreacted alkyne groups may remain in the conjugate, which may be capped using a suitable alkyne group capping agent, which in one aspect is an agent bearing an azide group.

[0336] In one embodiment, the alkyne group capping agent has the formula (VI): N3-X-OH (VI) wherein X is (CH) n and n is selected from 1 to 15. is a compound of

[0337] In one embodiment, the alkyne group capping agent is 3-azido-1-propanol. Thus, in one aspect, after step (c), the process further comprises capping any remaining unreacted alkyne groups in the conjugate with an alkyne group capping agent.

[0338] After conjugation to the carrier protein, the glycoconjugate can be purified (concentrated with respect to the amount of saccharide-protein conjugate) by a variety of techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration operations, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. Thus, in one aspect, the process for producing the glycoconjugates of the invention includes a step of purifying the glycoconjugate after it has been produced.

[0339] In one aspect, the serotype 3 glycoconjugates of the invention are produced according to click chemistry as disclosed above and in application PCT / IB2022 / 054914, which is incorporated herein by reference in its entirety. Thus, in one embodiment, the serotype 3 glycoconjugates of the invention comprise a serotype trisaccharide covalently conjugated to a carrier protein (CP) via a spacer and have the general formula (VII):

[0340] [ka]

[0341] [Wherein X is CH2(CH2) n’ , (CH2CH2O) m CH2CH2, NHCO(CH2) n’ , NHCO(CH2CH2O) m CH2CH2, OCH2(CH2) n and O(CH2CH2O) m CH2CH2, n' is selected from 1 to 10, m is selected from 1 to 4, and X' is CH2O(CH2) n’’ CH2C=O, CH2O(CH2CH2O)m’ (CH2) n’’ CH2C=O, n'' is selected from 0 to 10, and m' is selected from 0 to 4. It has.

[0342] Formula (VII) is a schematic representation of a serotype 3 glycoconjugate of the invention. Linkages should not be understood to exist for every saccharide repeat unit. Rather, the majority of the S. pneumoniae serotype trisaccharide repeat units remain unmodified, with covalent linkages between the carrier protein and the saccharide for a minority of the saccharide repeat units. Furthermore, an individual carrier protein (CP) molecule may be linked to more than one S. pneumoniae serotype trisaccharide molecule, and an individual S. pneumoniae serotype trisaccharide molecule may be linked to more than one individual carrier protein (CP) molecule.

[0343] In a preferred embodiment, the serotype 3 glycoconjugate of the invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is CH2(CH2) n’ where n' is 2 and X' is CH2O(CH2) n’’ CH2C=O and n'' is 1.

[0344] In one aspect, the serotype 3 glycoconjugate of the invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is CH2(CH2) n’ and n' is selected from 1 to 10, and X' is CH2O(CH2) n’’CH2C=O and n" is selected from 0 to 10. In one embodiment, n' is selected from 1 to 5 and n" is selected from 0 to 10. In one embodiment, n' is selected from 1 to 5 and n" is selected from 0 to 5. In one embodiment, n' is selected from 1 to 3 and n" is selected from 0 to 3. In one embodiment, n' is selected from 1 to 2 and n" is selected from 0 to 2. In a particular embodiment, n' is 1 and n" is 0. In another embodiment, n' is 2 and n" is 0.

[0345] In one aspect, the serotype 3 glycoconjugate of the invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is CH2(CH2) n’ and n' is selected from 1 to 10, and CH2O(CH2CH2O) m’ (CH2) n’’ CH2C=O, n'' is selected from 0 to 10, and m' is selected from 0 to 4.

[0346] In one aspect, the serotype 3 glycoconjugate of the invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is (CH2CHO) m CH2CH2, m is selected from 1 to 4, and X' is CH2O(CH2) n’’ CH2C=O and n'' is selected from 0 to 10. In one embodiment, m is selected from 1 to 3 and n'' is selected from 0 to 10.

[0347] In one aspect, the serotype 3 glycoconjugate of the invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is (CH2CHO) m CH2CH2, m is selected from 1 to 4, and X' is CH2O(CH2CH2O) m’ (CH2) n’’CH2C=O, n'' is selected from 0 to 10, and m' is selected from 0 to 4.

[0348] In one aspect, the serotype 3 glycoconjugate of the invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is NHCO(CH) n’ and n' is selected from 1 to 10, and X' is CH2O(CH2) n’’ CH2C=O, and n'' is selected from 0 to 10.

[0349] In one aspect, the serotype 3 glycoconjugate of the invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is NHCO(CH) n’ and n' is selected from 1 to 10, and X' is CH2O(CH2CH2O) m’ (CH2) n’’ CH2C=O, n'' is selected from 0 to 10, and m' is selected from 0 to 4.

[0350] In one aspect, the serotype 3 glycoconjugate of the invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is NHCO(CHCHO) m CH2CH2, m is selected from 1 to 4, and X' is CH2O(CH2) n’’ CH2C=O and n'' is selected from 0 to 10. In one embodiment, m is selected from 1 to 3 and n'' is selected from 0 to 10.

[0351] In one aspect, the serotype 3 glycoconjugate of the invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is NHCO(CHCHO) m CH2CH2, m is selected from 1 to 4, and X' is CH2O(CH2CH2O)m’ (CH2) n’’ CH2C=O, n'' is selected from 0 to 10, and m' is selected from 0 to 4.

[0352] In one aspect, the serotype 3 glycoconjugate of the invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is OCH2(CH2) n’ and n' is selected from 1 to 10, and X' is CH2O(CH2) n’’ CH2C=O, and n'' is selected from 0 to 10.

[0353] In one aspect, the serotype 3 glycoconjugate of the invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is OCH2(CH2) n’ and n' is selected from 1 to 10, and X' is CH2O(CH2CH2O) m’ (CH2) n’’ CH2C=O, n'' is selected from 0 to 10, and m' is selected from 0 to 4.

[0354] In one aspect, the serotype 3 glycoconjugate of the invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is O(CH2CHO) m CH2CH2, m is selected from 1 to 4, and X' is CH2O(CH2) n’’ CH2C=O, and n'' is selected from 0 to 10.

[0355] In one aspect, the serotype 3 glycoconjugate of the invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is O(CH2CHO) m CH2CH2, m is selected from 1 to 4, and X' is CH2O(CH2CH2O) m’ (CH2)n’’ CH2C=O, n'' is selected from 0 to 10, and m' is selected from 0 to 4.

[0356] After conjugation to a carrier protein, the serotype 3 glycoconjugates can be purified (concentrated with respect to the amount of saccharide-protein conjugate) by a variety of techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration operations, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. Thus, in one aspect, the process for producing the glycoconjugates of the invention includes a step of purifying the glycoconjugates after they have been produced. 1.7 Streptococcus pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 15B, 14, 18C, 19A, 19F, 22F, 23F and 33F glycoconjugates of the present invention In one aspect, the invention relates to a composition comprising a Streptococcus pneumoniae serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F glycoconjugate.

[0357] The structures of Streptococcus pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F polysaccharides are known in the art (see, e.g., Geno K et al. (2015) Clin Microbiol Rev 28:3, 871-899).

[0358] In one aspect, the Streptococcus pneumoniae serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F capsular saccharide used in the invention is a synthetic carbohydrate. Preparation of synthetic Streptococcus pneumoniae type 1 capsular saccharide can be carried out, for example, as disclosed in WO2015004041. Preparation of synthetic Streptococcus pneumoniae type 4 capsular saccharide can be carried out, for example, as disclosed in WO2016091399. Preparation of synthetic Streptococcus pneumoniae type 5 capsular saccharide can be carried out, for example, as disclosed in WO2016198170. Preparation of synthetic Streptococcus pneumoniae type 8 capsular saccharide can be carried out, for example, as disclosed in WO2017220753.

[0359] However, in a preferred embodiment, the source of bacterial polysaccharides according to the present invention may be Streptococcus pneumoniae bacterial cells. Bacterial strains that can be used as a source of Streptococcus pneumoniae capsular polysaccharides can be obtained from established culture collections (such as, for example, from the Streptococcus Reference Laboratory (US Centers for Disease Control and Prevention, Atlanta, GA, USA)) or from clinical specimens.

[0360] Streptococcus pneumoniae serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F capsular polysaccharides can be obtained directly from the bacteria using isolation procedures known to those skilled in the art. They can also be purchased commercially, for example from the American Type Culture Collection (ATCC), Manassas, Virginia, USA (e.g., reference numbers ATCC 13-X, ATCC 36-X, ATCC 41-X, ATCC 280-X, ATCC 107-X, ATCC 284-X, ATCC 505-X, ATCC 331-X, ATCC 511-X, ATCC 514-X, ATCC 517-X, ATCC 520-X, ATCC 81-X, ATCC 289-X, ATCC 304-X, ATCC 101-X, ATCC 527-X, ATCC 104-X, ATCC 535-X).

[0361] When capsular polysaccharides are obtained directly from bacteria, they can be grown in bacterial cell culture media, preferably in soy-based media. After fermentation of the bacterial cells that produce pneumococcal capsular polysaccharides, the bacterial cells can be lysed to produce a cell lysate. The capsular polysaccharides can then be isolated from the cell lysate using purification techniques known in the art, including centrifugation, depth filtration, precipitation, ultrafiltration, treatment with activated carbon, diafiltration and / or column chromatography (see, for example, US2006 / 0228380, US2006 / 0228381, WO2008 / 118752 and WO2020170190). The purified capsular polysaccharides can then be used to prepare glycoconjugates.

[0362] The isolated capsular saccharides may be characterised by a variety of parameters including, for example, weight average molecular weight (Mw). The molecular weight of the capsular saccharides can be measured by size exclusion chromatography (SEC) coupled with a multi-angle laser light scattering detector (MALLS).

[0363] In preferred embodiments, the isolated capsular polysaccharide (i.e., purified prior to further processing) has a weight average molecular weight of between 5 kDa and 5,000 kDa. In one embodiment, the isolated capsular polysaccharide has a weight average molecular weight of between 10 kDa and 3,000 kDa. In one embodiment, the isolated capsular polysaccharide has a weight average molecular weight of between 50 kDa and 1,000 kDa.

[0364] Any integer within any of the above ranges is contemplated as an aspect of the present disclosure. To produce conjugates with advantageous filterability, immunogenicity, and / or yield, capsular polysaccharides can be sized to a target molecular weight range prior to conjugation to a carrier protein. Advantageously, the size of purified serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and / or 33F capsular polysaccharides is reduced while retaining important structural features of the polysaccharide. Mechanical or chemical sizing can be used (see, for example, WO2006 / 110381, WO2015110941).

[0365] In one aspect, the size of the purified capsular polysaccharide is reduced by chemical hydrolysis. Chemical hydrolysis can be carried out using a weak acid (e.g., acetic acid, formic acid, propanoic acid). Chemical hydrolysis can also be carried out using a dilute strong acid (e.g., dilute hydrochloric acid, dilute sulfuric acid, dilute phosphoric acid, dilute nitric acid, or dilute perchloric acid).

[0366] The size of purified polysaccharides can also be reduced by mechanical homogenization. In one aspect, the size of purified polysaccharides is reduced by high-pressure homogenization, which achieves high shear rates by pumping the process stream through channels of sufficiently small dimensions. The shear rate can be increased by using greater applied homogenization pressure, and the exposure time can be increased by recirculating the feed stream through the homogenizer.

[0367] In one aspect the isolated serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F capsular polysaccharide is unsized.

[0368] The isolated serotype 1 capsular polysaccharide may be de-O-acetylated (see, e.g., WO2008 / 079653). Thus, in one embodiment, the isolated serotype 1 capsular polysaccharide is partially de-O-acetylated. In one embodiment, de-O-acetylation is carried out using a mild base. Partial de-O-acetylation may be carried out using a sodium bicarbonate / carbonate buffer.

[0369] In one aspect, the isolated serotype 1 capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1000 kDa. In one aspect, the isolated serotype 1 capsular polysaccharide prior to conjugation has a weight average molecular weight of between 150 kDa and 900 kDa. In a preferred aspect, the isolated serotype 1 capsular polysaccharide prior to conjugation has a weight average molecular weight of between 150 kDa and 700 kDa. The weight average molecular weight (Mw) of the isolated saccharide prior to conjugation refers to the Mw prior to activation of the polysaccharide (i.e., after the final sizing step, but before reacting the polysaccharide with an activating agent).

[0370] In one aspect, the isolated serotype 4 capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1000 kDa. In one aspect, the isolated serotype 4 capsular polysaccharide prior to conjugation has a weight average molecular weight of between 300 kDa and 900 kDa.

[0371] In one aspect, the isolated serotype 5 capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1,000 kDa. In one aspect, the isolated serotype 5 capsular polysaccharide prior to conjugation has a weight average molecular weight of between 200 kDa and 600 kDa.

[0372] In one aspect, the isolated serotype 6A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1000 kDa. In one aspect, the isolated serotype 6A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 300 kDa and 900 kDa.

[0373] In one aspect, the isolated serotype 6B capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1000 kDa, hi one aspect, the isolated serotype 6B capsular polysaccharide prior to conjugation has a weight average molecular weight of between 200 kDa and 900 kDa.

[0374] In one aspect, the isolated serotype 7F capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1000 kDa, hi one aspect, the isolated serotype 7F capsular polysaccharide prior to conjugation has a weight average molecular weight of between 200 kDa and 900 kDa.

[0375] In one aspect, the isolated serotype 8 capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1000 kDa. In one aspect, the isolated serotype 8 capsular polysaccharide prior to conjugation has a weight average molecular weight of between 200 kDa and 400 kDa.

[0376] In one aspect, the isolated serotype 9V capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1000 kDa. In one aspect, the isolated serotype 9V capsular polysaccharide prior to conjugation has a weight average molecular weight of between 200 kDa and 900 kDa.

[0377] In one aspect, the isolated serotype 10A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1000 kDa. In one aspect, the isolated serotype 10A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 200 kDa and 900 kDa.

[0378] In one aspect, the isolated serotype 11A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1000 kDa. In one aspect, the isolated serotype 11A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 400 kDa.

[0379] In one aspect, the isolated serotype 12F capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1000 kDa. In one aspect, the isolated serotype 12F capsular polysaccharide prior to conjugation has a weight average molecular weight of between 150 kDa and 400 kDa.

[0380] In one aspect, the isolated serotype 14 capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1000 kDa. In one aspect, the isolated serotype 14 capsular polysaccharide prior to conjugation has a weight average molecular weight of between 200 kDa and 900 kDa.

[0381] In one aspect, the isolated serotype 15B capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1000 kDa. In one aspect, the isolated serotype 15B capsular polysaccharide prior to conjugation has a weight average molecular weight of between 150 kDa and 300 kDa.

[0382] In one aspect, the isolated serotype 18C capsular polysaccharide prior to conjugation has a weight average molecular weight of between 20 kDa and 1000 kDa. In one aspect, the isolated serotype 18C capsular polysaccharide prior to conjugation has a weight average molecular weight of between 20 kDa and 500 kDa.

[0383] In one aspect, the isolated serotype 19A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1000 kDa. In one aspect, the isolated serotype 19A capsular polysaccharide prior to conjugation has a weight average molecular weight of between 250 kDa and 700 kDa.

[0384] In one aspect, the isolated serotype 19F capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1000 kDa. In one aspect, the isolated serotype 19F capsular polysaccharide prior to conjugation has a weight average molecular weight of between 250 kDa and 800 kDa.

[0385] In one aspect, the isolated serotype 22F capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1000 kDa, hi one aspect, the isolated serotype 22F capsular polysaccharide prior to conjugation has a weight average molecular weight of between 400 kDa and 700 kDa.

[0386] In one aspect, the isolated serotype 23F capsular polysaccharide prior to conjugation has a weight average molecular weight of between 100 kDa and 1000 kDa. In one aspect, the isolated serotype 23F capsular polysaccharide prior to conjugation has a weight average molecular weight of between 200 kDa and 800 kDa.

[0387] In one aspect, the isolated serotype 33F capsular polysaccharide prior to conjugation has a weight average molecular weight of between 300 kDa and 2000 kDa. In one aspect, the isolated serotype 33F capsular polysaccharide prior to conjugation has a weight average molecular weight of between 500 kDa and 2000 kDa.

[0388] In one embodiment, the serotype 1 glycoconjugate of the invention comprises a serotype 1 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 200 kDa and 750 kDa. In a preferred embodiment, the weight average molecular weight (Mw) is between 250 kDa and 600 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0389] In one aspect, the serotype 4 glycoconjugate of the invention comprises a serotype 4 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one aspect, the weight average molecular weight (Mw) is between 200 kDa and 1,000 kDa. In a preferred aspect, the weight average molecular weight (Mw) is between 400 kDa and 900 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0390] In one aspect, the serotype 5 glycoconjugate of the invention comprises a serotype 5 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 100 kDa and 1,000 kDa. In one aspect, the weight average molecular weight (Mw) is between 150 kDa and 800 kDa. In a preferred aspect, the weight average molecular weight (Mw) is between 200 kDa and 500 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0391] In one aspect, the serotype 6A glycoconjugate of the invention comprises a serotype 6A capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one aspect, the weight average molecular weight (Mw) is between 200 kDa and 1,000 kDa. In a preferred aspect, the weight average molecular weight (Mw) is between 300 kDa and 800 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0392] In one embodiment, the serotype 6B glycoconjugate of the invention comprises a serotype 6B capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 200 kDa and 1,000 kDa. In a preferred embodiment, the weight average molecular weight (Mw) is between 300 kDa and 800 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0393] In one embodiment, the serotype 7F glycoconjugate of the invention comprises a serotype 7F capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 200 kDa and 1,000 kDa. In a preferred embodiment, the weight average molecular weight (Mw) is between 300 kDa and 800 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0394] In one embodiment, the serotype 8 glycoconjugate of the invention comprises a serotype 8 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 200 kDa and 800 kDa. In a preferred embodiment, the weight average molecular weight (Mw) is between 300 kDa and 600 kDa. In a most preferred embodiment, the weight average molecular weight (Mw) is between 200 kDa and 400 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0395] In one embodiment, the serotype 9V glycoconjugate of the invention comprises a serotype 9V capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 200 kDa and 900 kDa. In a preferred embodiment, the weight average molecular weight (Mw) is between 300 kDa and 600 kDa. In a most preferred embodiment, the weight average molecular weight (Mw) is between 100 kDa and 400 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0396] In one embodiment, the serotype 10A glycoconjugate of the invention comprises a serotype 10A capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 200 kDa and 800 kDa. In a preferred embodiment, the weight average molecular weight (Mw) is between 300 kDa and 600 kDa. In a most preferred embodiment, the weight average molecular weight (Mw) is between 100 kDa and 400 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0397] In one aspect, the serotype 11A glycoconjugate of the invention comprises a serotype 11A capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one aspect, the weight average molecular weight (Mw) is between 75 kDa and 600 kDa. In a preferred aspect, the weight average molecular weight (Mw) is between 100 kDa and 400 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0398] In one embodiment, the serotype 12F glycoconjugate of the present invention comprises a serotype 12F capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 100 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 150 kDa and 600 kDa. In a preferred embodiment, the weight average molecular weight (Mw) is between 150 kDa and 400 kDa. In a most preferred embodiment, the weight average molecular weight (Mw) is between 250 kDa and 350 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0399] In one aspect, the serotype 14 glycoconjugate of the invention comprises a serotype 14 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one aspect, the weight average molecular weight (Mw) is between 100 kDa and 800 kDa. In a preferred aspect, the weight average molecular weight (Mw) is between 200 kDa and 600 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0400] In one aspect, the serotype 15B glycoconjugate of the invention comprises a serotype 15B capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one aspect, the weight average molecular weight (Mw) is between 100 kDa and 600 kDa. In a preferred aspect, the weight average molecular weight (Mw) is between 150 kDa and 300 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0401] In one aspect, the serotype 18C glycoconjugate of the invention comprises a serotype 18C capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 20 kDa and 800 kDa. In one aspect, the weight average molecular weight (Mw) is between 20 kDa and 400 kDa. In a preferred aspect, the weight average molecular weight (Mw) is between 20 kDa and 200 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0402] In one aspect, the serotype 19A glycoconjugate of the invention comprises a serotype 19A capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one aspect, the weight average molecular weight (Mw) is between 100 kDa and 700 kDa. In a preferred aspect, the weight average molecular weight (Mw) is between 250 kDa and 500 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0403] In one aspect, the serotype 19F glycoconjugate of the invention comprises a serotype 19F capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one aspect, the weight average molecular weight (Mw) is between 100 kDa and 900 kDa. In a preferred aspect, the weight average molecular weight (Mw) is between 250 kDa and 600 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0404] In one aspect, the serotype 22F glycoconjugate of the invention comprises a serotype 22F capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one aspect, the weight average molecular weight (Mw) is between 100 kDa and 900 kDa. In a preferred aspect, the weight average molecular weight (Mw) is between 400 kDa and 700 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0405] In one embodiment, the serotype 23F glycoconjugate of the invention comprises a serotype 23F capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 100 kDa and 900 kDa. In a preferred embodiment, the weight average molecular weight (Mw) is between 200 kDa and 600 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0406] In one embodiment, the serotype 33F glycoconjugate of the invention comprises a serotype 33F capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 2,500 kDa. In one embodiment, the weight average molecular weight (Mw) is between 100 kDa and 2,000 kDa. In a preferred embodiment, the weight average molecular weight (Mw) is between 600 kDa and 2,000 kDa. Here, the weight average molecular weight (Mw) prior to conjugation refers to the Mw after activation of the polysaccharide (i.e., after the final sizing step and after the polysaccharide has been reacted with an activating agent).

[0407] In some embodiments, the serotype 1 glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In other embodiments, the serotype 1 glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 10,000 kDa. In preferred embodiments, the serotype 1 glycoconjugates have a weight average molecular weight (Mw) between 2,000 kDa and 5,000 kDa.

[0408] In some embodiments, the serotype 4 glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In other embodiments, the serotype 4 glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 10,000 kDa. In preferred embodiments, the serotype 4 glycoconjugates have a weight average molecular weight (Mw) between 2,000 kDa and 5,000 kDa.

[0409] In some embodiments, the serotype 5 glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In other embodiments, the serotype 5 glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 10,000 kDa. In preferred embodiments, the serotype 5 glycoconjugates have a weight average molecular weight (Mw) between 2,000 kDa and 5,000 kDa.

[0410] In some embodiments, the serotype 6A glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In other embodiments, the serotype 6A glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 10,000 kDa. In preferred embodiments, the serotype 6A glycoconjugates have a weight average molecular weight (Mw) between 2,000 kDa and 5,000 kDa.

[0411] In some embodiments, the serotype 6B glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In other embodiments, the serotype 6B glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 10,000 kDa. In preferred embodiments, the serotype 6B glycoconjugates have a weight average molecular weight (Mw) between 2,000 kDa and 5,500 kDa.

[0412] In some embodiments, the serotype 7F glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In other embodiments, the serotype 7F glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 10,000 kDa. In preferred embodiments, the serotype 7F glycoconjugates have a weight average molecular weight (Mw) between 2,000 kDa and 5,500 kDa.

[0413] In some embodiments, the serotype 8 glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In other embodiments, the serotype 8 glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 10,000 kDa. In preferred embodiments, the serotype 8 glycoconjugates have a weight average molecular weight (Mw) between 2,500 kDa and 8,000 kDa.

[0414] In some embodiments, the serotype 9V glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In other embodiments, the serotype 9V glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 10,000 kDa. In preferred embodiments, the serotype 9V glycoconjugates have a weight average molecular weight (Mw) between 2,000 kDa and 5,500 kDa.

[0415] In some embodiments, the serotype 10A glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In other embodiments, the serotype 10A glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 10,000 kDa. In preferred embodiments, the serotype 10A glycoconjugates have a weight average molecular weight (Mw) between 2,000 kDa and 5,500 kDa.

[0416] In some embodiments, the serotype 11A glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In other embodiments, the serotype 11A glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 10,000 kDa. In preferred embodiments, the serotype 11A glycoconjugates have a weight average molecular weight (Mw) between 700 kDa and 4,500 kDa.

[0417] In some embodiments, the serotype 12F glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In other embodiments, the serotype 12F glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 10,000 kDa. In preferred embodiments, the serotype 12F glycoconjugates have a weight average molecular weight (Mw) between 1,500 kDa and 4,000 kDa.

[0418] In some embodiments, the serotype 14 glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In other embodiments, the serotype 14 glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 10,000 kDa. In preferred embodiments, the serotype 14 glycoconjugates have a weight average molecular weight (Mw) between 2,000 kDa and 5,500 kDa.

[0419] In some embodiments, the serotype 15B glycoconjugates of the present invention have a weight average molecular weight (Mw) between 1,000 kDa and 25,000 kDa. In other embodiments, the serotype 15B glycoconjugates have a weight average molecular weight (Mw) between 2,000 kDa and 20,000 kDa. In preferred embodiments, the serotype 15B glycoconjugates have a weight average molecular weight (Mw) between 5,000 kDa and 15,000 kDa.

[0420] In some embodiments, the serotype 18C glycoconjugates of the present invention have a weight average molecular weight (Mw) between 150 kDa and 15,000 kDa. In other embodiments, the serotype 18C glycoconjugates have a weight average molecular weight (Mw) between 250 kDa and 7,000 kDa. In preferred embodiments, the serotype 18C glycoconjugates have a weight average molecular weight (Mw) between 300 kDa and 4,000 kDa.

[0421] In some embodiments, the serotype 19A glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In other embodiments, the serotype 19A glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 10,000 kDa. In preferred embodiments, the serotype 19A glycoconjugates have a weight average molecular weight (Mw) between 2,000 kDa and 7,500 kDa.

[0422] In some embodiments, the serotype 19F glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In other embodiments, the serotype 19F glycoconjugates have a weight average molecular weight (Mw) between 750 kDa and 10,000 kDa. In preferred embodiments, the serotype 19F glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 7,500 kDa.

[0423] In some embodiments, the serotype 22F glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 10,000 kDa. In other embodiments, the serotype 22F glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 7,500 kDa. In preferred embodiments, the serotype 22F glycoconjugates have a weight average molecular weight (Mw) between 2,500 kDa and 5,500 kDa.

[0424] In some embodiments, the serotype 23F glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In other embodiments, the serotype 23F glycoconjugates have a weight average molecular weight (Mw) between 750 kDa and 10,000 kDa. In preferred embodiments, the serotype 23F glycoconjugates have a weight average molecular weight (Mw) between 1,000 kDa and 7,500 kDa.

[0425] In some embodiments, the serotype 33F glycoconjugates of the present invention have a weight average molecular weight (Mw) between 500 kDa and 15,000 kDa. In other embodiments, the serotype 33F glycoconjugates have a weight average molecular weight (Mw) between 750 kDa and 10,000 kDa. In preferred embodiments, the serotype 33F glycoconjugates have a weight average molecular weight (Mw) between 2,000 kDa and 6,000 kDa.

[0426] The glycoconjugates of the present invention can be characterized by the saccharide to carrier protein ratio (weight / weight). In some embodiments, the ratio of serotype 1 polysaccharide to carrier protein (w / w) in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.6 and 2.0. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0427] In some embodiments, the ratio (w / w) of serotype 4 polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.9 and 2.1. Even more preferably, the saccharide to carrier protein ratio (w / w) is between 1.0 and 1.9. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0428] In some embodiments, the ratio (w / w) of serotype 5 polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 1.3 and 2.5. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0429] In some embodiments, the ratio (w / w) of serotype 6A polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.7 and 1.6. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0430] In some embodiments, the ratio (w / w) of serotype 6B polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.4 and 0.8. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0431] In some embodiments, the ratio (w / w) of serotype 7F polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.7 and 1.5. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM.197 is.

[0432] In some embodiments, the ratio (w / w) of serotype 8 polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.6 and 1.6. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0433] In some embodiments, the ratio (w / w) of serotype 9V polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 1.2 and 2.3. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0434] In some embodiments, the ratio (w / w) of serotype 10A polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.7 and 1.6. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0435] In some embodiments, the ratio (w / w) of serotype 11A polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.9 and 1.5. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0436] In some embodiments, the ratio (w / w) of serotype 12 F polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.7 and 1.5. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0437] In some embodiments, the ratio (w / w) of serotype 14 polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 1.4 and 2.6. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0438] In some embodiments, the ratio (w / w) of serotype 15B polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.6 and 1.6. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0439] In some embodiments, the ratio (w / w) of serotype 18C polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.7 and 1.5. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0440] In some embodiments, the ratio (w / w) of serotype 19A polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.4 and 0.9. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0441] In some embodiments, the ratio (w / w) of serotype 19F polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.5 and 1.0. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0442] In some embodiments, the ratio (w / w) of serotype 22F polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.8 and 1.2. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0443] In some embodiments, the ratio (w / w) of serotype 23F polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 0.4 and 1.0. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0444] In some embodiments, the ratio (w / w) of serotype 33F polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the saccharide to carrier protein ratio (w / w) is between 1.0 and 2.2. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 is.

[0445] Another method for characterizing the glycoconjugates of the present invention is by the number of lysine residues in the carrier protein (e.g., CRM197, DT, or TT) that become conjugated to the saccharide, which can be characterized as the extent of conjugated lysines (degree of conjugation). Evidence of lysine modification of the carrier protein by covalent linkage to the polysaccharide can be obtained by amino acid analysis using routine methods known to those skilled in the art. Conjugation results in a reduction in the number of recovered lysine residues compared to the carrier protein starting material used to generate the conjugate material.

[0446] In a preferred embodiment, the degree of conjugation of the serotype 1 glycoconjugates of the invention is between 2 and 15. In a preferred embodiment, the degree of conjugation of the serotype 4 glycoconjugates of the present invention is between 2 and 15.

[0447] In a preferred embodiment, the degree of conjugation of the serotype 5 glycoconjugates of the invention is between 2 and 15. In a preferred embodiment, the degree of conjugation of the serotype 6A glycoconjugates of the present invention is between 2 and 15.

[0448] In a preferred embodiment, the degree of conjugation of the serotype 6B glycoconjugates of the present invention is between 2 and 15. In a preferred embodiment, the degree of conjugation of the serotype 7F glycoconjugates of the invention is between 2 and 15.

[0449] In a preferred embodiment, the degree of conjugation of the serotype 8 glycoconjugates of the present invention is between 2 and 15. In a preferred embodiment, the degree of conjugation of the serotype 9V glycoconjugates of the present invention is between 2 and 15.

[0450] In a preferred embodiment, the degree of conjugation of the serotype 10A glycoconjugates of the present invention is between 2 and 15. In a preferred embodiment, the degree of conjugation of the serotype 11A glycoconjugates of the present invention is between 2 and 15.

[0451] In a preferred embodiment, the degree of conjugation of the serotype 12F glycoconjugates of the present invention is between 2 and 15. In a preferred embodiment, the degree of conjugation of the serotype 14 glycoconjugates of the invention is between 2 and 15.

[0452] In a preferred embodiment, the degree of conjugation of the serotype 15B glycoconjugates of the present invention is between 2 and 15. In a preferred embodiment, the degree of conjugation of the serotype 18C glycoconjugates of the present invention is between 2 and 15.

[0453] In a preferred embodiment, the degree of conjugation of the serotype 19A glycoconjugates of the present invention is between 2 and 15. In a preferred embodiment, the degree of conjugation of the serotype 19F glycoconjugates of the present invention is between 2 and 15.

[0454] In a preferred embodiment, the degree of conjugation of the serotype 22F glycoconjugates of the present invention is between 2 and 15. In a preferred embodiment, the degree of conjugation of the serotype 23F glycoconjugates of the present invention is between 2 and 15.

[0455] In a preferred embodiment, the degree of conjugation of the serotype 33F glycoconjugates of the present invention is between 2 and 15. Serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, or 33F glycoconjugates of the invention and immunogenic compositions comprising said glycoconjugate(s) may contain free saccharides that are not covalently conjugated to a carrier protein but are nevertheless present in the glycoconjugate composition. The free saccharides may be non-covalently associated with (i.e., non-covalently bound to, adsorbed to, or entrapped in or with) the glycoconjugate.

[0456] In one embodiment, the serotype 1 glycoconjugates of the invention comprise less than about 40% free serotype 1 polysaccharide relative to the total amount of serotype 1 polysaccharide. In a preferred embodiment, the serotype 1 glycoconjugates comprise less than about 20% free serotype 1 polysaccharide relative to the total amount of serotype 1 polysaccharide.

[0457] In one embodiment, the serotype 1 glycoconjugates of the invention comprise less than about 40% free serotype 1 polysaccharide relative to the total amount of serotype 1 polysaccharide. In a preferred embodiment, the serotype 1 glycoconjugates comprise less than about 20% free serotype 1 polysaccharide relative to the total amount of serotype 1 polysaccharide.

[0458] In one embodiment, the serotype 4 glycoconjugates of the invention comprise less than about 40% free serotype 4 polysaccharide relative to the total amount of serotype 4 polysaccharide. In a preferred embodiment, the serotype 4 glycoconjugates comprise less than about 30% free serotype 4 polysaccharide relative to the total amount of serotype 4 polysaccharide.

[0459] In one embodiment, the serotype 5 glycoconjugates of the invention comprise less than about 45% free serotype 5 polysaccharide relative to the total amount of serotype 5 polysaccharide. In a preferred embodiment, the serotype 5 glycoconjugates comprise less than about 40% free serotype 5 polysaccharide relative to the total amount of serotype 5 polysaccharide.

[0460] In one embodiment, the serotype 6A glycoconjugates of the invention comprise less than about 40% free serotype 6A polysaccharide relative to the total amount of serotype 6A polysaccharide. In a preferred embodiment, the serotype 6A glycoconjugates comprise less than about 30% free serotype 6A polysaccharide relative to the total amount of serotype 6A polysaccharide.

[0461] In one embodiment, the serotype 6B glycoconjugates of the present invention comprise less than about 30% free serotype 6B polysaccharide relative to the total amount of serotype 6B polysaccharide. In a preferred embodiment, the serotype 6B glycoconjugates comprise less than about 20% free serotype 6B polysaccharide relative to the total amount of serotype 6B polysaccharide.

[0462] In one embodiment, the serotype 7F glycoconjugates of the invention comprise less than about 30% free serotype 7F polysaccharide relative to the total amount of serotype 7F polysaccharide. In a preferred embodiment, the serotype 7F glycoconjugates comprise less than about 20% free serotype 7F polysaccharide relative to the total amount of serotype 7F polysaccharide.

[0463] In one embodiment, the serotype 8 glycoconjugates of the invention comprise less than about 30% free serotype 8 polysaccharide relative to the total amount of serotype 8 polysaccharide. In a preferred embodiment, the serotype 8 glycoconjugates comprise less than about 20% free serotype 8 polysaccharide relative to the total amount of serotype 8 polysaccharide.

[0464] In one embodiment, the serotype 9V glycoconjugates of the invention comprise less than about 40% free serotype 9V polysaccharide relative to the total amount of serotype 9V polysaccharide. In a preferred embodiment, the serotype 9V glycoconjugates comprise less than about 35% free serotype 9V polysaccharide relative to the total amount of serotype 9V polysaccharide.

[0465] In one embodiment, the serotype 10A glycoconjugates of the invention comprise less than about 40% free serotype 10A polysaccharide relative to the total amount of serotype 10A polysaccharide. In a preferred embodiment, the serotype 10A glycoconjugates comprise less than about 20% free serotype 10A polysaccharide relative to the total amount of serotype 10A polysaccharide.

[0466] In one embodiment, the serotype 11A glycoconjugate of the present invention comprises less than about 40% free serotype 11A polysaccharide relative to the total amount of serotype 11A polysaccharide. In a preferred embodiment, the serotype 11A glycoconjugate comprises less than about 30% free serotype 11A polysaccharide relative to the total amount of serotype 11A polysaccharide.

[0467] In one embodiment, the serotype 12F glycoconjugate of the present invention comprises less than about 40% free serotype 12F polysaccharide relative to the total amount of serotype 12F polysaccharide. In a preferred embodiment, the serotype 12F glycoconjugate comprises less than about 30% free serotype 12F polysaccharide relative to the total amount of serotype 12F polysaccharide.

[0468] In one embodiment, the serotype 14 glycoconjugates of the invention comprise less than about 40% free serotype 14 polysaccharide relative to the total amount of serotype 14 polysaccharide. In a preferred embodiment, the serotype 14 glycoconjugates comprise less than about 35% free serotype 14 polysaccharide relative to the total amount of serotype 14 polysaccharide.

[0469] In one embodiment, the serotype 15B glycoconjugates of the present invention comprise less than about 40% free serotype 15B polysaccharide relative to the total amount of serotype 15B polysaccharide. In a preferred embodiment, the serotype 15B glycoconjugates comprise less than about 35% free serotype 15B polysaccharide relative to the total amount of serotype 15B polysaccharide.

[0470] In one embodiment, the serotype 18C glycoconjugate of the present invention comprises less than about 30% free serotype 18C polysaccharide relative to the total amount of serotype 18C polysaccharide. In a preferred embodiment, the serotype 18C glycoconjugate comprises less than about 20% free serotype 18C polysaccharide relative to the total amount of serotype 18C polysaccharide.

[0471] In one embodiment, the serotype 19A glycoconjugates of the invention comprise less than about 40% free serotype 19A polysaccharide relative to the total amount of serotype 19A polysaccharide. In a preferred embodiment, the serotype 19A glycoconjugates comprise less than about 30% free serotype 19A polysaccharide relative to the total amount of serotype 19A polysaccharide.

[0472] In one embodiment, the serotype 19F glycoconjugate of the present invention comprises less than about 30% free serotype 19F polysaccharide relative to the total amount of serotype 19F polysaccharide. In a preferred embodiment, the serotype 19F glycoconjugate comprises less than about 20% free serotype 19F polysaccharide relative to the total amount of serotype 19F polysaccharide.

[0473] In one embodiment, the serotype 22F glycoconjugates of the invention comprise less than about 40% free serotype 22F polysaccharide relative to the total amount of serotype 22F polysaccharide. In a preferred embodiment, the serotype 22F glycoconjugates comprise less than about 20% free serotype 22F polysaccharide relative to the total amount of serotype 22F polysaccharide.

[0474] In one embodiment, the serotype 23F glycoconjugates of the invention comprise less than about 30% free serotype 23F polysaccharide relative to the total amount of serotype 23F polysaccharide. In a preferred embodiment, the serotype 23F glycoconjugates comprise less than about 20% free serotype 22F polysaccharide relative to the total amount of serotype 23F polysaccharide.

[0475] In one embodiment, the serotype 33F glycoconjugates of the invention comprise less than about 30% free serotype 33F polysaccharide relative to the total amount of serotype 33F polysaccharide. In a preferred embodiment, the serotype 33F glycoconjugates comprise less than about 20% free serotype 33F polysaccharide relative to the total amount of serotype 33F polysaccharide.

[0476] Serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, or 33F glycoconjugates of the present invention can be characterized by molecular size distribution (Kd). Size exclusion chromatography media (CL-4B) can be used to determine the relative molecular size distribution of the conjugate. Size exclusion chromatography (SEC) is used in a gravity-fed column to profile the molecular size distribution of the conjugate. Large molecules excluded from the pores in the media elute more rapidly than small molecules. A fraction collector is used to collect the column eluate. Fractions are tested colorimetrically by a saccharide assay. To determine Kd, the column is calibrated to establish the fraction at which molecules are completely excluded (V0), (Kd=0), and the fraction representing maximum retention (Vi), (Kd=1). The fraction (Ve) at which a particular sample attribute is reached is related to Kd by the expression Kd = (Ve - V0) / (Vi - V0).

[0477] In one embodiment, at least 40% of the serotype 1 glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 50% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 50% and 80% of the serotype 1 glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0478] In one embodiment, at least 30% of the serotype 4 glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 40% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 40% and 80% of the serotype 4 glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0479] In one embodiment, at least 40% of the serotype 5 glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 55% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 55% and 80% of the serotype 5 glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0480] In one embodiment, at least 50% of the serotype 6A glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 60% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 60% and 90% of the serotype 6A glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0481] In one embodiment, at least 30% of the serotype 6B glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 35% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 35% and 80% of the serotype 6B glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0482] In one embodiment, at least 50% of the serotype 7F glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 65% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 65% and 90% of the serotype 7F glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0483] In one embodiment, at least 60% of the serotype 8 glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 70% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 70% and 90% of the serotype 8 glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0484] In one embodiment, at least 30% of the serotype 9V glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 40% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 40% and 80% of the serotype 9V glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0485] In one embodiment, at least 40% of the serotype 10A glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 55% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 55% and 85% of the serotype 10A glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0486] In one embodiment, at least 50% of the serotype 11A glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 60% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 60% and 85% of the serotype 11A glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0487] In one embodiment, at least 40% of the serotype 12F glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 50% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 50% and 80% of the serotype 12F glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0488] In one embodiment, at least 40% of the serotype 14 glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 50% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 50% and 80% of the serotype 14 glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0489] In one embodiment, at least 30% of the serotype 15B glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 40% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 40% and 80% of the serotype 15B glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0490] In one embodiment, at least 30% of the serotype 18C glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 40% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 40% and 80% of the serotype 18C glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0491] In one embodiment, at least 40% of the serotype 19A glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 50% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 50% and 80% of the serotype 19A glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0492] In one embodiment, at least 40% of the serotype 19F glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 50% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 50% and 80% of the serotype 19F glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0493] In one embodiment, at least 30% of the serotype 22F glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 40% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 40% and 80% of the serotype 22F glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0494] In one embodiment, at least 30% of the serotype 23F glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 35% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 35% and 80% of the serotype 23F glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0495] In one embodiment, at least 50% of the serotype 33F glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, at least 60% of the glycoconjugates have a Kd of 0.3 or less in a CL-4B column. In a preferred embodiment, between 60% and 95% of the serotype 33F glycoconjugates have a Kd of 0.3 or less in a CL-4B column.

[0496] In one embodiment, serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and / or 33F saccharides are activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form cyanate esters. The activated polysaccharides are then coupled, either directly or via a spacer (linker) group, to amino groups on a carrier protein. For example, the spacer can be cystamine or cysteamine, resulting in a thiolated polysaccharide, which can be coupled to the carrier by a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyrloxy] succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g., using iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl) aminobenzoate (SlAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA), or succinimidyl 3-[bromoacetamido] propionate (SBAP)). Preferably, the cyanate ester is coupled with hexanediamine or adipic acid dihydrazide (ADH), and the amino-derivatized saccharide is attached to the carrier protein (e.g., CRM) using carbodiimide (e.g., EDAC or EDC) chemistry via carboxyl groups on the protein carrier. 197 Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348 and WO 96 / 129094.

[0497] Other suitable techniques for conjugation use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU. Many are described in International Patent Application Publication No. WO 98 / 42721. Conjugation may involve a carbonyl linker, which can be formed by reaction of the free hydroxyl group of a sugar with 1,1'-carbonyldiimidazole (CDI) (see Bethell et al. (1979) J. Biol. Chern. 254:2572-2574; Hearn et al. (1981) J. Chromatogr. 218:509-518), followed by reaction with the protein to form a carbamate bond. This may involve reducing the anomeric terminus to a primary hydroxyl group, optionally protecting / deprotecting the primary hydroxyl group, reacting the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling the CDI carbamate intermediate to an amino group on the protein.

[0498] In one aspect, serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F glycoconjugates of the invention are prepared using reductive amination chemistry. According to the present invention, reductive amination involves two steps: (1) oxidizing (activating) purified saccharides, and (2) reducing the activated saccharides and carrier proteins to form glycoconjugates (see, e.g., WO2006 / 110381, WO2008 / 079653, WO2008 / 143709, WO2008 / 079732, WO2011 / 110531, WO2012 / 119972, WO2015110941, WO2015110940, WO2018 / 144439, WO2018 / 156491).

[0499] In one aspect, serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F glycoconjugates of the invention are prepared using reductive amination chemistry.

[0500] In another embodiment, serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F and 23F glycoconjugates of the invention are prepared using reductive amination chemistry.

[0501] As described above, sizing of isolated serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and / or 33F capsular polysaccharides to a target molecular weight (MW) range may be performed prior to oxidation. Thus, in one aspect, isolated serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and / or 33F capsular polysaccharides are sized prior to oxidation.

[0502] Thus, in one aspect, the isolated serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F capsular polysaccharides are sized prior to oxidation.

[0503] In one aspect, the isolated serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F capsular polysaccharide is conjugated to a carrier protein by a process comprising the following steps: (a) reacting the isolated serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F capsular polysaccharide with an oxidizing agent to produce an activated polysaccharide; (b) combining the activated polysaccharide of step (a) with a carrier protein; and (c) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate.

[0504] The saccharides are said to be activated after the oxidation step (a) and are called "activated polysaccharides". In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes terminal hydroxyl groups to aldehydes. In one embodiment, the oxidizing agent is periodate. For purposes of the present invention, the term "periodate" includes both periodate and periodic acid, and the term also includes metaperiodate (IO 4- ) and orthoperiodate (IO6 5- ) and various salts of periodate (e.g., sodium periodate and potassium periodate).

[0505] In one aspect, isolated serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F capsular polysaccharides are reacted with periodate.

[0506] In one aspect, isolated serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F and 23F capsular polysaccharides are reacted with periodate.

[0507] In a preferred embodiment, the oxidizing agent is sodium periodate. In one embodiment, the periodate used for oxidation is metaperiodate. In a most preferred embodiment, the periodate used for oxidation is sodium metaperiodate.

[0508] When a polysaccharide is reacted with periodate, the periodate oxidizes vicinal hydroxyl groups to form carbonyl or aldehyde groups, causing cleavage of C-C bonds. For this reason, the term "reacting a polysaccharide with periodate" includes the oxidation of vicinal hydroxyl groups by periodate.

[0509] In one aspect, step a) comprises reacting serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F polysaccharide with periodate.

[0510] In one aspect, step a) comprises reacting serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F polysaccharide with 0.05 to 2 molar equivalents of periodate.

[0511] In one aspect, step a) comprises reacting serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F polysaccharide with 0.05 to 0.2 molar equivalents of periodate.

[0512] In one aspect, step a) comprises reacting serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F polysaccharide with 0.2 to 0.5 molar equivalents of periodate.

[0513] In one aspect, step a) comprises reacting serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F polysaccharide with 0.5 to 1.5 molar equivalents of periodate.

[0514] In one aspect, step a) comprises reacting serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F polysaccharide with 1.5 to 2.0 molar equivalents of periodate.

[0515] In some aspects, the reaction of step (a) is quenched. Thus, after step (a), a quenching step (a') may be performed (see WO2015110940). Thus, in one aspect, isolated serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F capsular polysaccharide is conjugated to a carrier protein by a process comprising the following steps: (a) reacting the isolated serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F capsular polysaccharide with an oxidizing agent to produce an activated polysaccharide; (a') quenching the oxidation reaction by adding a quenching agent to result in the activated polysaccharide; (b) combining the activated polysaccharide of step (a') with a carrier protein; and (c) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate.

[0516] In one embodiment, the oxidizing agent is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical and N-chlorosuccinimide (NCS) as a co-oxidant. This oxidizing agent is particularly suitable for oxidizing serotype 12F capsular polysaccharides. In such an embodiment, glycoconjugates from Streptococcus pneumoniae serotype 12F are prepared using 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical to oxidize primary alcohols of sugars to aldehydes using N-chlorosuccinimide (NCS) as a co-oxidant (hereinafter "TEMPO / NCS oxidation"), e.g., as described in Example 7 and in WO 2014 / 097099. Thus, in one aspect, glycoconjugates from Streptococcus pneumoniae serotype 12F of the present invention can be obtained by a method (hereinafter referred to as "TEMPO / NCS-reductive amination") comprising the following steps: (a) reacting isolated serotype 12F capsular polysaccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) to produce an activated polysaccharide, (b) combining the activated polysaccharide of step (a) with a carrier protein, and (c) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate. In one aspect, glycoconjugates from Streptococcus pneumoniae serotype 12F can be obtained by the method.

[0517] In one embodiment, isolated serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F capsular polysaccharides are reacted with periodate and isolated serotype 12F capsular polysaccharide is reacted with TEMPO / NCS.

[0518] In one embodiment, isolated serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F and 23F capsular polysaccharides are reacted with periodate and isolated serotype 12F capsular polysaccharide is reacted with TEMPO / NCS.

[0519] In preferred embodiments, the degree of oxidation (also termed "degree of activation" herein) of the activated serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F polysaccharides is between 2 and 30.

[0520] In one embodiment, when the carrier protein is TT, the degree of oxidation of the activated serotype 1 polysaccharide is between 1 and 15 (see, for example, Table 2 of WO2019 / 152921). In a preferred embodiment, the carrier protein is a CRM 197 When the degree of oxidation of the activated serotype 1 polysaccharide is between 4 and 10, see Table 1 of WO2019 / 152921.

[0521] In one embodiment, the degree of oxidation of the activated serotype 4 polysaccharide is between 1 and 15. In a preferred embodiment, the carrier protein is a CRM 197 , the degree of oxidation of the activated serotype 4 polysaccharide is between 1 and 5. See Table 1 of WO2019 / 152921.

[0522] In one embodiment, the degree of oxidation of the activated serotype 5 polysaccharide is between 1 and 15. In a preferred embodiment, the carrier protein is a CRM 197 , the degree of oxidation of the activated serotype 5 polysaccharide is between 2 and 6. See Table 1 of WO2019 / 152921.

[0523] In one embodiment, when the carrier protein is TT, the degree of oxidation of the activated serotype 5 polysaccharide is between 1 and 15. See Table 2 of WO2019 / 152921. In one embodiment, the degree of oxidation of the activated serotype 6A polysaccharide is between 1 and 15. In a preferred embodiment, the carrier protein is a CRM 197 When the degree of oxidation of the activated serotype 6A polysaccharide is between 5 and 15. See Table 1 of WO2019 / 152921.

[0524] In one embodiment, the degree of oxidation of the activated serotype 6B polysaccharide is between 1 and 15. In a preferred embodiment, the carrier protein is a CRM 197 When the degree of oxidation of the activated serotype 6B polysaccharide is between 7 and 13, see Table 1 of WO2019 / 152921.

[0525] In one embodiment, the degree of oxidation of the activated serotype 7F polysaccharide is between 1 and 15. In a preferred embodiment, the carrier protein is a CRM 197 , the degree of oxidation of the activated serotype 7F polysaccharide is between 2 and 8. See Table 1 of WO2019 / 152921.

[0526] In one embodiment, the degree of oxidation of the activated serotype 8 polysaccharide is between 1 and 20. In a preferred embodiment, the carrier protein is a CRM 197 , the degree of oxidation of the activated serotype 8 polysaccharide is between 1 and 17. See Table 1 of WO2019 / 152921.

[0527] In one embodiment, the degree of oxidation of the activated serotype 9V polysaccharide is between 1 and 15. In a preferred embodiment, the carrier protein is a CRM 197 , the degree of oxidation of the activated serotype 9V polysaccharide is between 4 and 9. See Table 1 of WO2019 / 152921.

[0528] In one embodiment, the degree of oxidation of the activated serotype 10A polysaccharide is between 1 and 15. In a preferred embodiment, the carrier protein is a CRM 197 , the degree of oxidation of the activated serotype 10A polysaccharide is between 1 and 12. See Table 1 of WO2019 / 152921.

[0529] In one embodiment, the degree of oxidation of the activated serotype 11A polysaccharide is between 1 and 20. In a preferred embodiment, the carrier protein is a CRM 197 , the degree of oxidation of the activated serotype 11A polysaccharide is between 1 and 15. See Table 1 of WO2019 / 152921.

[0530] In one embodiment, the degree of oxidation of the activated serotype 12 F polysaccharide is between 1 and 15. In a preferred embodiment, the carrier protein is a CRM 197 , the degree of oxidation of the activated serotype 12 F polysaccharide is between 1 and 9. See Table 1 of WO2019 / 152921.

[0531] In one embodiment, the degree of oxidation of the activated serotype 14 polysaccharide is between 1 and 20. In a preferred embodiment, the carrier protein is a CRM 197 When the degree of oxidation of the activated serotype 14 polysaccharide is between 6 and 13, see Table 1 of WO2019 / 152921.

[0532] In one embodiment, the degree of oxidation of the activated serotype 15B polysaccharide is between 1 and 20. In a preferred embodiment, the carrier protein is a CRM 197 When the degree of oxidation of the activated serotype 15B polysaccharide is between 1 and 17. See Table 1 of WO2019 / 152921.

[0533] In one embodiment, when the carrier protein is TT, the degree of oxidation of the activated serotype 15B polysaccharide is between 1 and 15. See Table 2 of WO2019 / 152925. In one embodiment, the degree of oxidation of the activated serotype 18C polysaccharide is between 1 and 20. In a preferred embodiment, the carrier protein is a CRM 197 When the degree of oxidation of the activated serotype 18C polysaccharide is between 6 and 14. See Table 1 of WO2019 / 152921.

[0534] In one embodiment, the degree of oxidation of the activated serotype 19A polysaccharide is between 1 and 20. In a preferred embodiment, the carrier protein is a CRM 197 When the degree of oxidation of the activated serotype 19A polysaccharide is between 7 and 13, see Table 1 of WO2019 / 152921.

[0535] In one embodiment, the degree of oxidation of the activated serotype 19F polysaccharide is between 1 and 20. In a preferred embodiment, the carrier protein is a CRM 197When the degree of oxidation of the activated serotype 19F polysaccharide is between 6 and 12. See Table 1 of WO2019 / 152921.

[0536] In one embodiment, the degree of oxidation of the activated serotype 22F polysaccharide is between 1 and 20. In a preferred embodiment, the carrier protein is a CRM 197 , the degree of oxidation of the activated serotype 22F polysaccharide is between 1 and 16. See Table 1 of WO2019 / 152921.

[0537] In one embodiment, when the carrier protein is TT, the degree of oxidation of the activated serotype 22F polysaccharide is between 1 and 20. See Table 2 of WO2019 / 152925. In one embodiment, the degree of oxidation of the activated serotype 23F polysaccharide is between 1 and 20. In a preferred embodiment, the carrier protein is a CRM 197 , the degree of oxidation of the activated serotype 23F polysaccharide is between 6 and 14. See Table 1 of WO2019 / 152921.

[0538] In one embodiment, the degree of oxidation of the activated serotype 33F polysaccharide is between 1 and 20. In a preferred embodiment, the carrier protein is a CRM 197 , the degree of oxidation of the activated serotype 33F polysaccharide is between 1 and 15. See Table 1 of WO2019 / 152921.

[0539] The activated polysaccharide and carrier protein can be lyophilized (freeze-dried) independently (separate lyophilization) or together (co-lyophilized). In one embodiment, the activated polysaccharide and carrier protein are co-lyophilized. In another embodiment, the activated polysaccharide and carrier protein are lyophilized independently.

[0540] In one embodiment, lyophilization occurs in the presence of a non-reducing sugar, possible non-reducing sugars including sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, and palatinit.

[0541] In one aspect, the initial weight ratio of activated serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F capsular polysaccharide to carrier protein in step b) is between 4: 1 and 0.1: 1. In one aspect, the initial weight ratio of activated serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F capsular polysaccharide to carrier protein in step b) is between 1.5: 1 and 0.5: 1.

[0542] In one embodiment, the reduction reaction (c) is carried out in an aprotic solvent. In one embodiment, the reduction reaction (c) is carried out in a solution consisting essentially of dimethyl sulfoxide (DMSO). In one embodiment, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) solvent.

[0543] In one embodiment, the reduction reaction (c) is carried out in an aqueous solvent. In one embodiment, for serotypes 6A, 6B, 7F, 8, 10A, 15B, 19A, 19F, 22F and 23F, reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) solvent, and for serotypes 1, 4, 5, 9V, 11A, 12F, 14 and 18C, reduction reaction (c) is carried out in an aqueous solvent.

[0544] In one embodiment, for serotypes 6A, 6B, 7F, 8, 10A, 15B, 19A, 19F, 22F and 23F, reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) solvent, and for serotypes 1, 4, 5, 9V, 11A, 12F, 14, 18C and 33F, reduction reaction (c) is carried out in an aqueous solvent.

[0545] In one embodiment, for serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F, reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) solvent, and for serotypes 1, 4, 5, 9V, 14, 22F and 33F, reduction reaction (c) is carried out in an aqueous solvent.

[0546] In one embodiment, for serotypes 6A, 6B, 7F, 11A, 12F, 19A, 19F and 23F, reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) solvent, and for serotypes 1, 4, 5, 8, 9V, 10A, 14, 15B, 18C, 22F and 33F, reduction reaction (c) is carried out in an aqueous solvent.

[0547] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium or zinc borohydride, amine borane, such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3, or 5-ethyl-2-methylpyridine borane (PEMB), in the presence of a Bronsted acid or Lewis acid. In one embodiment, the reducing agent is sodium triacetoxyborohydride. In a preferred embodiment, the reducing agent is sodium cyanoborohydride. In one embodiment, the reducing agent is sodium cyanoborohydride in the presence of nickel (see WO2018144439).

[0548] In one embodiment, between 0.2 and 10 molar equivalents of reducing agent are used in step c), preferably between 0.5 and 5 molar equivalents of reducing agent are used in step c).

[0549] At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugate, which can be capped using a suitable capping agent, which in one aspect is sodium borohydride (NaBH4).

[0550] In one aspect, capping is achieved by combining the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In one aspect, capping is achieved by combining the product of step c) with 1 to 3 molar equivalents of sodium borohydride.

[0551] After conjugation to a carrier protein, serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and / or 33F glycoconjugates can be purified (concentrated with respect to the amount of saccharide-protein conjugate) by a variety of techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration operations, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. Thus, in one aspect, a process for producing a serotype 1 glycoconjugate of the invention includes a step of purifying the glycoconjugate after it has been produced.

[0552] In certain embodiments, serotype 33F glycoconjugates of the invention are prepared using reductive amination. In other embodiments, serotype 33F glycoconjugates of the invention are prepared using eTEC conjugation (hereinafter "serotype 33F eTEC-linked glycoconjugates") as described in WO2014 / 027302 or WO2015110941 (see Examples 1, 2, and 3). The 33F glycoconjugates comprise saccharides covalently conjugated to a carrier protein via one or more eTEC spacers, wherein the saccharides are covalently conjugated to the eTEC spacer via a carbamate bond and the carrier protein is covalently conjugated to the eTEC spacer via an amide bond. The eTEC-linked glycoconjugates of the invention have the general formula (III):

[0553] [ka]

[0554] [wherein the atoms comprising the eTEC spacer are contained in the central box] It can be expressed as: The eTEC spacer contains seven linear atoms (i.e., -C(O)NH(CH2)2SCH2C(O)-) and provides stable thioether and amide bonds between the saccharide and the carrier protein. Synthesis of eTEC-linked glycoconjugates involves reacting the activated hydroxyl group of the saccharide with the amino group of a thioalkylamine reagent, e.g., cystamine or cysteine ​​amine or a salt thereof, to form a carbamate bond to the saccharide, providing a thiolated saccharide. Generation of one or more free sulfhydryl groups is achieved by reaction with a reducing agent to provide an activated thiolated saccharide. Reaction of the free sulfhydryl group of the activated thiolated saccharide with an activated carrier protein bearing one or more α-haloacetamide groups on the amine-containing residue generates a thioether bond, forming a conjugate in which the carrier protein is attached to the eTEC spacer via an amide bond.

[0555] In the serotype 33F eTEC-linked glycoconjugates of the present invention, the saccharide can be a polysaccharide or an oligosaccharide. The carrier protein can be selected from any suitable carrier described herein or known to those of skill in the art. Preferably, the saccharide is a polysaccharide. In some such embodiments, the carrier protein is a CRM. 197 In some such embodiments, the eTEC-linked glycoconjugate comprises Streptococcus pneumoniae serotype 33F capsular polysaccharide.

[0556] In particularly preferred embodiments, the eTEC-linked glycoconjugate is linked to a CRM via an eTEC spacer. 197 (serotype 33F eTEC-linked glycoconjugate).

[0557] In one aspect, serotype 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F and 23F glycoconjugates of the invention are prepared using reductive amination chemistry, and serotype 33F glycoconjugates of the invention are prepared using eTEC conjugation. 1.8 Carrier Proteins of the Invention A component of the glycoconjugates of the present invention is a carrier protein to which a saccharide is conjugated. The terms "protein carrier" or "carrier protein" or "carrier" may be used interchangeably herein. The carrier protein must be suitable for standard conjugation procedures. Capsular saccharides may be conjugated to the carrier protein via covalent or non-covalent bonds. In one aspect, the capsular saccharide is conjugated to the carrier protein via a non-covalent bond (e.g., the resavidin / biotin system, see, e.g., WO2012155007, WO2020056202). Preferably, the capsular saccharide is conjugated via a covalent bond.

[0558] In one embodiment, the carrier protein of the glycoconjugate of the present invention is DT (diphtheria toxoid), TT (tetanus toxoid) or fragment C of TT, CRM 197 (a non-toxic but antigenically identical variant of diphtheria toxin), other DT mutants (e.g., CRM 176 , CRM 228 , CRM 45 (Uchida et al. (1973) J. Biol. Chem. 218:3838-3844), CRM9, CRM 102 , CRM 103 or CRM 107and Nicholls and Youle in Genetically Engineered Toxins, Eds: Frankel, Maecel Dekker Other mutations described by Inc. (1992); deletion of Glu-148 or mutation to Asp, Gln or Ser and / or mutation of Ala158 to GIy and other mutations disclosed in U.S. Pat. Nos. 4,709,017 and 4,950,740, mutation of at least one or more of residues Lys516, Lys526, Phe530 and / or Lys534 and other mutations disclosed in U.S. Pat. Nos. 5,917,017 and 6,455,673; or fragments disclosed in U.S. Pat. No. 5,843,711, ply detoxified in some way, for example, dPLY-GMBS (WO 2004 / 081515, WO 2006 / 032499) or coccoccal pneumolysin (ply) containing dPLY-formol (Kuo et al. (1995) Infect Immun 63:2706-2713), PhtX, including PhtA, PhtB, PhtD, PhtE (the sequences of PhtA, PhtB, PhtD or PhtE are disclosed in WO00 / 37105 and WO00 / 39299), and fusions of Pht proteins, such as PhtDE fusions, PhtBE fusions, PhtAE (WO01 / 98334, WO03 / 054007, WO2009 / 000826), OMPC (meningococcal outer membrane protein) commonly extracted from Neisseria meningitidis serovars (EP0372501), PorB (from Neisseria meningitidis), PD (Haemophilus influenzae protein D;see, for example, EP 0 594 610 B), or immunologically functional equivalents thereof, synthetic peptides (EP 0 378 881 , EP 0 427 347 ), heat shock proteins (WO 93 / 17712 , WO 94 / 03208 ), pertussis proteins (WO 98 / 58668 , EP 0 471 177 ), cytokines, lymphokines, growth factors or hormones (WO 91 / 01146 ), artificial proteins containing multiple human CD4+ T cell epitopes derived from antigens from various pathogens (Falugi et al. (2001) Eur J Immunol 31:3816-3824), e.g., N19 protein (Baraldoi et al. (2004) Infect Immunol 72: pp. 4884-4887) pneumococcal surface protein PspA (WO02 / 091998), iron uptake protein (WO01 / 72337), Clostridium difficile toxin A or B (WO00 / 61761), transferrin-binding protein, pneumococcal adhesion protein (PsaA) or recombinant Pseudomonas aeruginosa aeruginosa exotoxin A (particularly non-toxic mutants thereof, such as exotoxin A with a substitution at glutamic acid 553 (Douglas et al. (1987) J. Bacteriol. 169(11):4967-4971)). Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD), can also be used as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins, such as cholera toxoid (e.g., as described in WO 2004 / 083251), Escherichia coli toxin (E. coli), and the like. Exotoxin A from Escherichia coli (E. coli) LT, E. coli (E. coli) ST, and P. aeruginosa (P. aeruginosa) is another suitable carrier protein. C5a peptidase (SCP) from Streptococcus is another suitable carrier protein.

[0559] In one embodiment, the carrier protein of a glycoconjugate of the invention is the fusion protein CP1. The CP1 fusion protein comprises a biotin-binding protein, such as, for example, a truncated resavidin protein (e.g., amino acids 45-179 of a wild-type resavidin protein), a first linker (e.g., a GGGSSSS linker), an SP1500 polypeptide (e.g., amino acids 27-278 of a full-length Streptococcus pneumoniae SP1500 polypeptide), a second linker (e.g., amino acid sequence AAA), and an SP0785 polypeptide (e.g., amino acids 33-399 of a full-length Streptococcus pneumoniae SP0785 polypeptide), see, e.g., WO2020056202.

[0560] In another embodiment, the carrier protein of the glycoconjugate of the invention is PD (H. influenzae protein D; see, eg, EP0594610B).

[0561] Preferably, the carrier protein of the glycoconjugate of the present invention is DT, TT, CRM 197 or C5a peptidase (SCP) derived from Streptococcus genus. In one embodiment, the carrier protein of the glycoconjugate of the invention is DT (diphtheria toxoid).

[0562] In another embodiment, the carrier protein of the glycoconjugate of the present invention is TT (tetanus toxoid). In a preferred embodiment, the carrier protein of the glycoconjugate of the present invention is a CRM 197 or C5a peptidase (SCP) derived from Streptococcus genus.

[0563] In a preferred embodiment, the carrier protein of the glycoconjugate of the present invention is a CRM 197 CRM 197 The protein is a non-toxic form of diphtheria toxin, but is immunologically indistinguishable from diphtheria toxin. 197The atoxic phage β197 was generated by nitrosoguanidine mutagenesis of the toxigenic corynephage β. tox- CRM is produced by Corynebacterium diphtheriae infected with HIV (Uchida et al. (1971) Nature New Biology 233:8-11). 197 The protein has the same molecular weight as diphtheria toxin but differs from it by a single base change (guanine to adenine) in the structural gene, which causes an amino acid substitution (glutamic acid to glycine) in the mature protein, eliminating the toxicity of diphtheria toxin. 197 Proteins are safe and effective T-cell dependent carriers of saccharides. 197 and further details regarding its production can be found, for example, in US Pat. No. 5,614,382.

[0564] In one aspect, the carrier protein of the glycoconjugate of the invention is a CRM 197 (See CN103495161). In one embodiment, the carrier protein of the glycoconjugate of the present invention is a CRM obtained by expression in recombinant Escherichia coli. 197 (See CN103495161).

[0565] In another preferred embodiment, the carrier protein of the glycoconjugate of the present invention is SCP (Streptococcus C5a peptidase). Methods have been developed to overcome this immune response in two important species of β-hemolytic streptococci, Streptococcus pyogenes (Group A Streptococcus, GAS) and Streptococcus agalactiae (Group B Streptococcus, GBS), which cause a variety of serious human infections ranging from mild cases of pharyngitis and impetigo to severe invasive diseases such as necrotizing fasciitis (GAS) and neonatal sepsis (GBS). All human isolates of β-hemolytic streptococci, including GAS and GBS, produce a highly conserved cell wall protein, SCP (Streptococcus C5a peptidase), which specifically inactivates C5a. The scp genes from GAS and GBS encode polypeptides containing between 1,134 and 1,181 amino acids (Brown et al., PNAS, 2005, Vol. 102, No. 51, pp. 18391-18396). The first 31 residues are a trafficking signal presequence, which is removed during passage through the cell membrane. The next 68 residues act as a prosequence and must be removed to yield an active SCP. The next 10 residues can be removed without loss of protease activity. At the other end, beginning with Lys-1034, there are four consecutive 17-residue motifs, followed by a cell sorting and cell wall attachment signal. This combined signal consists of a 20-residue hydrophilic sequence containing the LPTTND sequence, a 17-residue hydrophobic sequence, and a short basic carboxyl terminus.

[0566] SCPs can be divided into domains (see Figure 1B in Brown et al., PNAS, 2005, Vol. 102, No. 51, pp. 18391-18396). These domains include the pre / pro domain (which contains the trafficking signal presequence (typically the first 31 residues) and the prosequence (typically the next 68 residues)), the protease domain (divided into two parts: protease part 1, typically residues 89-333 / 334, and protease domain part 2, typically residues 467 / 468-583 / 584), and the protease-associated domain (PA domain). (generally, residues 333 / 334 to 467 / 468), three fibronectin type III (Fn) domains (Fn1, generally, residues 583 / 584 to 712 / 713; Fn2, generally, residues 712 / 713 to 928 / 929 / 930; Fn3, generally, residues 929 / 930 to 1029 / 1030 / 1031), and a cell wall anchor domain (generally, residues 1029 / 1030 / 1031 at the C-terminus).

[0567] In one embodiment, the carrier protein of the glycoconjugate of the invention is SCP (SCPB) from GBS. An example of SCPB is provided in SEQ ID NO: 3 of WO 97 / 26008. See also SEQ ID NO: 3 of WO 00 / 34487.

[0568] In another preferred embodiment, the carrier protein of the glycoconjugate of the present invention is a GAS-derived SCP (SCPA). Examples of SCPA can be found in SEQ ID NO: 1 and SEQ ID NO: 2 of WO 97 / 26008. See also SEQ ID NO: 1, 2 and 23 of WO 00 / 34487.

[0569] In a preferred embodiment, the carrier protein of the glycoconjugates of the present invention is an enzymatically inactive SCP. In another preferred embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive GBS-derived SCP (SCPB).

[0570] In another preferred embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive GAS-derived SCP (SCPA). In one embodiment, the carrier protein of the glycoconjugate of the present invention is a fragment of SCP. In one embodiment, the carrier protein of the glycoconjugate of the present invention is a fragment of SCPA. Preferably, the carrier protein of the glycoconjugate of the present invention is a fragment of SCPB.

[0571] In one embodiment, the carrier protein of the glycoconjugate of the invention is a fragment of SCP that contains the protease domain, the protease-associated domain (PA domain) and three fibronectin type III (Fn) domains, but does not contain the transport signal pre-sequence, pro-sequence and cell wall anchor domain.

[0572] In one embodiment, the carrier protein of the glycoconjugate of the invention is a fragment of SCP that contains the protease domain, the protease-associated domain (PA domain) and three fibronectin type III (Fn) domains, but does not contain the transport signal pre-sequence, pro-sequence and cell wall anchor domain.

[0573] In one embodiment, the carrier protein of the glycoconjugates of the invention is an enzymatically inactive fragment of SCP that contains the protease domain, the protease-associated domain (PA domain), and two of the three fibronectin type III (Fn) domains, but does not contain the transport signal pre-sequence, pro-sequence, and cell wall anchor domain.

[0574] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP, which contains the protease domain, the protease-associated domain (PA domain), and the three fibronectin type III (Fn) domains, but does not contain the transport signal pre-sequence, the pro-sequence, and the cell wall anchor domain.

[0575] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of SCPA, which contains the protease domain, the protease-associated domain (PA domain), and the three fibronectin type III (Fn) domains, but does not contain the transport signal pre-sequence, the pro-sequence, and the cell wall anchor domain.

[0576] In a preferred embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of SCPB, preferably comprising the protease domain, the protease-associated domain (PA domain), and the three fibronectin type III (Fn) domains, but excluding the transport signal pre-sequence, the pro-sequence, and the cell wall anchor domain.

[0577] In one embodiment, the enzymatic activity of the SCP is inactivated by replacing at least one amino acid in the wild-type sequence. In one embodiment, the replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. Numbers indicate amino acid residue positions in the peptidase according to the numbering of SEQ ID NO: 1 of WO00 / 34487.

[0578] Thus, in one embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive SCP, wherein said inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, said at least one amino acid replacement is in the protease domain. In one embodiment, said at least one amino acid replacement is in part 1 of the protease domain. In one embodiment, said at least one amino acid replacement is in part 2 of the protease domain. In one embodiment, said replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, said replacement is S512A.

[0579] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive SCPA, and said inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, said at least one amino acid replacement is in the protease domain. In one embodiment, said at least one amino acid replacement is in part 1 of the protease domain. In one embodiment, said at least one amino acid replacement is in part 2 of the protease domain. In one embodiment, said replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, said replacement is S512A.

[0580] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive SCPB, and said inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, said at least one amino acid replacement is in the protease domain. In one embodiment, said at least one amino acid replacement is in part 1 of the protease domain. In one embodiment, said at least one amino acid replacement is in part 2 of the protease domain. In one embodiment, said replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, said replacement is S512A.

[0581] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP, and said inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, said at least one amino acid replacement is in the protease domain. In one embodiment, said at least one amino acid replacement is in part 1 of the protease domain. In one embodiment, said at least one amino acid replacement is in part 2 of the protease domain. In one embodiment, said replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, said replacement is S512A.

[0582] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of SCP comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but not the trafficking signal pre-sequence, the pro-sequence, and the cell wall anchor domain, wherein said inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, said at least one amino acid replacement is in the protease domain. In one embodiment, said at least one amino acid replacement is in portion 1 of the protease domain. In one embodiment, said at least one amino acid replacement is in portion 2 of the protease domain. In one embodiment, said replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, the substitution is S512A.

[0583] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of SCPA comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but not the transport signal pre-sequence, the pro-sequence, and the cell wall anchor domain, and said inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, said at least one amino acid replacement is in the protease domain. In one embodiment, said at least one amino acid replacement is in portion 1 of the protease domain. In one embodiment, said at least one amino acid replacement is in portion 2 of the protease domain. In one embodiment, said replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, the substitution is S512A.

[0584] In one embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of SCPB comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but not the transport signal pre-sequence, the pro-sequence, and the cell wall anchor domain, and said inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, said at least one amino acid replacement is in the protease domain. In one embodiment, said at least one amino acid replacement is in portion 1 of the protease domain. In one embodiment, said at least one amino acid replacement is in portion 2 of the protease domain. In one embodiment, said replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, the substitution is S512A.

[0585] In one embodiment, the enzymatic activity of the SCP is inactivated by replacing at least two amino acids of the wild-type sequence. In one embodiment, said at least two amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said at least two amino acid replacements are D130A and H193A. In one embodiment, said at least two amino acid replacements are D130A and N295A. In one embodiment, said at least two amino acid replacements are D130A and S512A. In one embodiment, said at least two amino acid replacements are H193A and N295A. In one embodiment, said at least two amino acid replacements are H193A and S512A. In one embodiment, said at least two amino acid replacements are N295A and S512A.

[0586] Thus, in one embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive SCP, wherein said inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, said replacement of at least two amino acids is in the protease domain. In one embodiment, said replacement of at least two amino acids is in part 1 of the protease domain. In one embodiment, said replacement of at least two amino acids is in part 2 of the protease domain. In one embodiment, said replacement of at least two amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement of at least two amino acids is D130A and H193A. In one embodiment, said replacement of at least two amino acids is D130A and N295A. Preferably, said replacement of at least two amino acids is D130A and S512A. In one embodiment, said at least two amino acid replacements are H193A and N295A. In one embodiment, said at least two amino acid replacements are H193A and S512A. In one embodiment, said at least two amino acid replacements are N295A and S512A.

[0587] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive SCPA, and said inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, said replacement of at least two amino acids is in the protease domain. In one embodiment, said replacement of at least two amino acids is in part 1 of the protease domain. In one embodiment, said replacement of at least two amino acids is in part 2 of the protease domain. In one embodiment, said replacement of at least two amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement of at least two amino acids is D130A and H193A. In one embodiment, said replacement of at least two amino acids is D130A and N295A. Preferably, said replacement of at least two amino acids is D130A and S512A. In one embodiment, said at least two amino acid replacements are H193A and N295A. In one embodiment, said at least two amino acid replacements are H193A and S512A. In one embodiment, said at least two amino acid replacements are N295A and S512A.

[0588] In one embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive SCPB, and said inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, said replacement of at least two amino acids is in the protease domain. In one embodiment, said replacement of at least two amino acids is in part 1 of the protease domain. In one embodiment, said replacement of at least two amino acids is in part 2 of the protease domain. In one embodiment, said replacement of at least two amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement of at least two amino acids is D130A and H193A. In one embodiment, said replacement of at least two amino acids is D130A and N295A. Preferably, said replacement of at least two amino acids is D130A and S512A. In one embodiment, said at least two amino acid replacements are H193A and N295A. In one embodiment, said at least two amino acid replacements are H193A and S512A. In one embodiment, said at least two amino acid replacements are N295A and S512A.

[0589] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP, and said inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, said replacement of at least two amino acids is in the protease domain. In one embodiment, said replacement of at least two amino acids is in part 1 of the protease domain. In one embodiment, said replacement of at least two amino acids is in part 2 of the protease domain. In one embodiment, said replacement of at least two amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement of at least two amino acids is D130A and H193A. In one embodiment, said replacement of at least two amino acids is D130A and N295A. Preferably, said replacement of at least two amino acids is D130A and S512A. In one embodiment, said at least two amino acid replacements are H193A and N295A. In one embodiment, said at least two amino acid replacements are H193A and S512A. In one embodiment, said at least two amino acid replacements are N295A and S512A.

[0590] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of SCP comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but not the transport signal pre-sequence, the pro-sequence, and the cell wall anchor domain, wherein said inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, said replacement of at least two amino acids is in the protease domain. In one embodiment, said replacement of at least two amino acids is in portion 1 of the protease domain. In one embodiment, said replacement of at least two amino acids is in portion 2 of the protease domain. In one embodiment, said replacement of at least two amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement of at least two amino acids is D130A and H193A. In one embodiment, the at least two amino acid replacements are D130A and N295A. Preferably, the at least two amino acid replacements are D130A and S512A. In one embodiment, the at least two amino acid replacements are H193A and N295A. In one embodiment, the at least two amino acid replacements are H193A and S512A. In one embodiment, the at least two amino acid replacements are N295A and S512A.

[0591] In one embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of SCPA comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but not the transport signal pre-sequence, the pro-sequence, and the cell wall anchor domain, and said inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, said replacement of at least two amino acids is in the protease domain. In one embodiment, said replacement of at least two amino acids is in portion 1 of the protease domain. In one embodiment, said replacement of at least one amino acid is in portion 2 of the protease domain. In one embodiment, said replacement of at least two amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement of at least two amino acids is D130A and H193A. In one embodiment, the at least two amino acid replacements are D130A and N295A. Preferably, the at least two amino acid replacements are D130A and S512A. In one embodiment, the at least two amino acid replacements are H193A and N295A. In one embodiment, the at least two amino acid replacements are H193A and S512A. In one embodiment, the at least two amino acid replacements are N295A and S512A.

[0592] In one embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of SCPB comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but not the transport signal pre-sequence, the pro-sequence, and the cell wall anchor domain, and said inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, said replacement of at least two amino acids is in the protease domain. In one embodiment, said replacement of at least two amino acids is in part 1 of the protease domain. In one embodiment, said replacement of at least two amino acids is in part 2 of the protease domain. In one embodiment, said replacement of at least two amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement of at least two amino acids is D130A and H193A. In one embodiment, the at least two amino acid replacements are D130A and N295A. Preferably, the at least two amino acid replacements are D130A and S512A. In one embodiment, the at least two amino acid replacements are H193A and N295A. In one embodiment, the at least two amino acid replacements are H193A and S512A. In one embodiment, the at least two amino acid replacements are N295A and S512A.

[0593] In one embodiment, the enzymatic activity of the SCP is inactivated by replacing at least three amino acids of the wild-type sequence. In one embodiment, said at least three amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said at least three amino acid replacements are D130A, H193A, and N295A. In one embodiment, said at least three amino acid replacements are D130A, H193A, and S512A. In one embodiment, said at least three amino acid replacements are D130A, N295A, and S512A. In one embodiment, said at least three amino acid replacements are H193A, N295A, and S512A.

[0594] Thus, in one embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive SCP, wherein said inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, said replacement of at least three amino acids is in the protease domain. In one embodiment, said replacement of at least three amino acids is in part 1 of the protease domain. In one embodiment, said replacement of at least three amino acids is in part 2 of the protease domain. In one embodiment, said replacement of at least three amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement of at least three amino acids is D130A, H193A, and N295A. In one embodiment, said replacement of at least three amino acids is D130A, H193A, and S512A. In one embodiment, said at least three amino acid substitutions are D130A, N295A and S512A, hi one embodiment, said at least three amino acid substitutions are H193A, N295A and S512A.

[0595] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive SCPA, and said inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, said replacement of at least three amino acids is in the protease domain. In one embodiment, said replacement of at least three amino acids is in portion 1 of the protease domain. In one embodiment, said replacement of at least three amino acids is in portion 2 of the protease domain. In one embodiment, said replacement of at least three amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement of at least three amino acids is D130A, H193A, and N295A. In one embodiment, said replacement of at least three amino acids is D130A, H193A, and S512A. In one embodiment, said at least three amino acid substitutions are D130A, N295A and S512A, hi one embodiment, said at least three amino acid substitutions are H193A, N295A and S512A.

[0596] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive SCPB, and said inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, said replacement of at least three amino acids is in the protease domain. In one embodiment, said replacement of at least three amino acids is in part 1 of the protease domain. In one embodiment, said replacement of at least three amino acids is in part 2 of the protease domain. In one embodiment, said replacement of at least three amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement of at least three amino acids is D130A, H193A, and N295A. In one embodiment, said replacement of at least three amino acids is D130A, H193A, and S512A. In one embodiment, said at least three amino acid substitutions are D130A, N295A and S512A, hi one embodiment, said at least three amino acid substitutions are H193A, N295A and S512A.

[0597] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP, and said inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, said replacement of at least three amino acids is in the protease domain. In one embodiment, said replacement of at least three amino acids is in part 1 of the protease domain. In one embodiment, said replacement of at least three amino acids is in part 2 of the protease domain. In one embodiment, said replacement of at least three amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement of at least three amino acids is D130A, H193A, and N295A. In one embodiment, said replacement of at least three amino acids is D130A, H193A, and S512A. In one embodiment, said at least three amino acid substitutions are D130A, N295A and S512A, hi one embodiment, said at least three amino acid substitutions are H193A, N295A and S512A.

[0598] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of SCP comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but not the trafficking signal pre-sequence, the pro-sequence, and the cell wall anchor domain, wherein said inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, said replacement of at least three amino acids is in the protease domain. In one embodiment, said replacement of at least three amino acids is in portion 1 of the protease domain. In one embodiment, said replacement of at least three amino acids is in portion 2 of the protease domain. In one embodiment, said replacement of at least three amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement of at least three amino acids is D130A, H193A, and N295A. In one embodiment, said at least three amino acid replacements are D130A, H193A and S512A. In one embodiment, said at least three amino acid replacements are D130A, N295A and S512A. In one embodiment, said at least three amino acid replacements are H193A, N295A and S512A.

[0599] In one embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of SCPA comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but not the transport signal pre-sequence, the pro-sequence, and the cell wall anchor domain, and said inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, said replacement of at least three amino acids is in the protease domain. In one embodiment, said replacement of at least three amino acids is in portion 1 of the protease domain. In one embodiment, said replacement of at least three amino acids is in portion 2 of the protease domain. In one embodiment, said replacement of at least three amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement of at least three amino acids is D130A, H193A, and N295A. In one embodiment, said at least three amino acid replacements are D130A, H193A and S512A. In one embodiment, said at least three amino acid replacements are D130A, N295A and S512A. In one embodiment, said at least three amino acid replacements are H193A, N295A and S512A.

[0600] In one embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of SCPB comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but not the transport signal pre-sequence, the pro-sequence, and the cell wall anchor domain, and said inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, said replacement of at least three amino acids is in the protease domain. In one embodiment, said replacement of at least three amino acids is in part 1 of the protease domain. In one embodiment, said replacement of at least three amino acids is in part 2 of the protease domain. In one embodiment, said replacement of at least three amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement of at least three amino acids is D130A, H193A, and N295A. In one embodiment, said at least three amino acid replacements are D130A, H193A and S512A. In one embodiment, said at least three amino acid replacements are D130A, N295A and S512A. In one embodiment, said at least three amino acid replacements are H193A, N295A and S512A.

[0601] In one embodiment, the enzymatic activity of the SCP is inactivated by replacing at least four amino acids of the wild-type sequence, hi one embodiment, the replacement of at least four amino acids is D130A, H193A, N295A and S512A.

[0602] Thus, in one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive SCP, said inactivation being achieved by replacing at least four amino acids of the wild-type sequence. Preferably, said replacement of at least four amino acids is in the protease domain. In one embodiment, said replacement of at least four amino acids is in part 1 of the protease domain. In one embodiment, said replacement of at least four amino acids is in part 2 of the protease domain. In one embodiment, said replacement of at least four amino acids is D130A, H193A, N295A, and S512A.

[0603] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive SCPA, and said inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, said replacement of at least four amino acids is in the protease domain. In one embodiment, said replacement of at least four amino acids is in part 1 of the protease domain. In one embodiment, said replacement of at least four amino acids is in part 2 of the protease domain. In one embodiment, said replacement of at least four amino acids is D130A, H193A, N295A, and S512A.

[0604] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive SCPB, and said inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, said replacement of at least four amino acids is in the protease domain. In one embodiment, said replacement of at least four amino acids is in part 1 of the protease domain. In one embodiment, said replacement of at least four amino acids is in part 2 of the protease domain. In one embodiment, said replacement of at least four amino acids is D130A, H193A, N295A, and S512A.

[0605] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of SCP, and said inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, said replacement of at least four amino acids is in the protease domain. In one embodiment, said replacement of at least four amino acids is in part 1 of the protease domain. In one embodiment, said replacement of at least four amino acids is in part 2 of the protease domain. In one embodiment, said replacement of at least four amino acids is D130A, H193A, N295A, and S512A.

[0606] In one embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of SCP comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but not the transport signal pre-sequence, the pro-sequence, and the cell wall anchor domain, wherein said inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, said replacement of at least four amino acids is in the protease domain. In one embodiment, said replacement of at least four amino acids is in portion 1 of the protease domain. In one embodiment, said replacement of at least four amino acids is in portion 2 of the protease domain. In one embodiment, said replacement of at least four amino acids is D130A, H193A, N295A, and S512A.

[0607] In one embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of SCPA comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but not the transport signal pre-sequence, the pro-sequence, and the cell wall anchor domain, and said inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, said replacement of at least four amino acids is in the protease domain. In one embodiment, said replacement of at least four amino acids is in part 1 of the protease domain. In one embodiment, said replacement of at least one amino acid is in part 2 of the protease domain. In one embodiment, said replacement of at least four amino acids is D130A, H193A, N295A, and S512A.

[0608] In one embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of SCPB comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but not the transport signal pre-sequence, the pro-sequence, and the cell wall anchor domain, and said inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, said replacement of at least four amino acids is in the protease domain. In one embodiment, said replacement of at least four amino acids is in part 1 of the protease domain. In one embodiment, said replacement of at least four amino acids is in part 2 of the protease domain. In one embodiment, said replacement of at least four amino acids is D130A, H193A, N295A, and S512A.

[0609] In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of SEQ ID NO:1. In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of SEQ ID NO:2.

[0610] [ka]

[0611] SEQ ID NO:1 is 950 amino acids in length.

[0612] [ka]

[0613] SEQ ID NO:2 is 949 amino acids in length. In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 90% identity to SEQ ID NO:1.

[0614] In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 95% identity to SEQ ID NO:1.

[0615] In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 99% identity to SEQ ID NO:1.

[0616] In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 99.5% identity to SEQ ID NO:1.

[0617] In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 99.8% identity to SEQ ID NO:1.

[0618] In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 99.85% identity to SEQ ID NO:1.

[0619] In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 90% identity to SEQ ID NO:2.

[0620] In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 95% identity to SEQ ID NO:2.

[0621] In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 99% identity to SEQ ID NO:2.

[0622] In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 99.5% identity to SEQ ID NO:2.

[0623] In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 99.8% identity to SEQ ID NO:2.

[0624] In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 99.85% identity to SEQ ID NO:2. 2. Immunogenic composition of the present invention 2.1 Combinations of Glycoconjugates of the Invention In one aspect, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention (eg, those disclosed in section 1 above).

[0625] Preferably, the number of different S. pneumoniae capsular saccharides may range from 1 different serotype (or 'v', valency) to 25 different serotypes (25v). If the protein carrier is the same for two or more saccharides in a composition, the saccharides may be conjugated to the same molecule of protein carrier (a carrier molecule having two or more different saccharides conjugated to it) [see, e.g., WO2020121159].

[0626] However, in preferred embodiments, the saccharides are each individually conjugated to different molecules of the protein carrier (each molecule of the protein carrier having only one type of saccharide conjugated to it), in which embodiment the capsular saccharides are said to be individually conjugated to the carrier protein.

[0627] In one aspect, the present invention relates to an immunogenic composition comprising 1 to 25 different glycoconjugates of the invention (as disclosed in section 1 above). In one aspect, the present invention relates to an immunogenic composition comprising 1 to 25 glycoconjugates from different serotypes of Streptococcus pneumoniae (1 to 25 pneumococcal conjugates) of the present invention.

[0628] In one aspect, the present invention relates to an immunogenic composition comprising one pneumococcal glycoconjugate of the present invention. In one aspect, the present invention relates to an immunogenic composition comprising two pneumococcal glycoconjugates of the invention.

[0629] In one aspect, the present invention relates to an immunogenic composition comprising three pneumococcal glycoconjugates of the invention. In one aspect, the present invention relates to an immunogenic composition comprising four pneumococcal glycoconjugates of the present invention.

[0630] In one aspect, the present invention relates to an immunogenic composition comprising five pneumococcal glycoconjugates of the present invention. In one aspect, the present invention relates to an immunogenic composition comprising ten pneumococcal glycoconjugates of the present invention.

[0631] In one aspect, the present invention relates to an immunogenic composition comprising 11 pneumococcal glycoconjugates of the present invention. In one aspect, the present invention relates to an immunogenic composition comprising 13 pneumococcal glycoconjugates of the present invention.

[0632] In one aspect, the present invention relates to an immunogenic composition comprising 15 pneumococcal glycoconjugates of the present invention. In one aspect, the present invention relates to an immunogenic composition comprising 19 pneumococcal glycoconjugates of the present invention.

[0633] In one aspect, the present invention relates to an immunogenic composition comprising 20 pneumococcal glycoconjugates of the present invention. In one aspect, the present invention relates to an immunogenic composition comprising 21 pneumococcal glycoconjugates of the present invention.

[0634] In one aspect, the present invention relates to an immunogenic composition comprising 22 pneumococcal glycoconjugates of the present invention. In one aspect, the present invention relates to an immunogenic composition comprising 23 pneumococcal glycoconjugates of the present invention.

[0635] In one aspect, the present invention relates to an immunogenic composition comprising 24 pneumococcal glycoconjugates of the present invention. In a preferred embodiment, the present invention relates to an immunogenic composition comprising 25 pneumococcal glycoconjugates of the present invention.

[0636] In one aspect, the invention relates to an immunogenic composition comprising a glycoconjugate derived from Streptococcus pneumoniae serotype 15A (e.g., those in section 1 above). In one aspect, the invention relates to an immunogenic composition comprising a glycoconjugate derived from Streptococcus pneumoniae serotype 23A (eg, those in section 1 above).

[0637] In one aspect, the invention relates to an immunogenic composition comprising a glycoconjugate derived from Streptococcus pneumoniae serotype 23B (eg, those in section 1 above). In one aspect, the invention relates to an immunogenic composition comprising a glycoconjugate derived from Streptococcus pneumoniae serotype 24F (eg, those in section 1 above).

[0638] In one aspect, the invention relates to an immunogenic composition comprising a glycoconjugate derived from Streptococcus pneumoniae serotype 35B (eg, those in section 1 above). In one aspect, the invention relates to immunogenic compositions comprising glycoconjugates derived from Streptococcus pneumoniae serotypes 15A and 23A (eg, those in section 1 above).

[0639] In one aspect, the invention relates to immunogenic compositions comprising glycoconjugates derived from Streptococcus pneumoniae serotypes 15A and 23B (eg, those in section 1 above). In one aspect, the invention relates to immunogenic compositions comprising glycoconjugates derived from Streptococcus pneumoniae serotypes 15A and 24F (eg, those in section 1 above).

[0640] In one aspect, the invention relates to immunogenic compositions comprising glycoconjugates derived from Streptococcus pneumoniae serotypes 15A and 35B (e.g., those in section 1 above). In one aspect, the invention relates to immunogenic compositions comprising glycoconjugates derived from Streptococcus pneumoniae serotypes 23A and 23B (eg, those in section 1 above).

[0641] In one aspect, the invention relates to immunogenic compositions comprising glycoconjugates derived from Streptococcus pneumoniae serotypes 23A and 24F (eg, those in section 1 above). In one aspect, the invention relates to immunogenic compositions comprising glycoconjugates derived from Streptococcus pneumoniae serotypes 23A and 35B (eg, those in section 1 above).

[0642] In one aspect, the invention relates to immunogenic compositions comprising glycoconjugates derived from Streptococcus pneumoniae serotypes 23B and 24F (eg, those in section 1 above). In one aspect, the invention relates to immunogenic compositions comprising glycoconjugates derived from Streptococcus pneumoniae serotypes 23B and 35B (eg, those in section 1 above).

[0643] In one aspect, the invention relates to immunogenic compositions comprising glycoconjugates derived from Streptococcus pneumoniae serotypes 24F and 35B (eg, those in section 1 above). In one aspect, the invention relates to immunogenic compositions comprising glycoconjugates derived from Streptococcus pneumoniae serotypes 15A, 23A and 23B (eg, those in section 1 above).

[0644] In one aspect, the invention relates to immunogenic compositions comprising glycoconjugates derived from Streptococcus pneumoniae serotypes 15A, 23A and 24F (eg, those in section 1 above). In one aspect, the invention relates to immunogenic compositions comprising glycoconjugates derived from St...

Claims

1. 1. An immunogenic composition comprising glycoconjugates derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and further comprising 1-5 glycoconjugates derived from Streptococcus pneumoniae serotypes 15A, 23A, 23B, 24F and / or 35B.

2. 2. The immunogenic composition of claim 1, which is a 21-, 22-, 23-, 24-, or 25-valent pneumococcal conjugate composition.

3. 2. The immunogenic composition of claim 1, comprising a glycoconjugate derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and 35B.

4. The immunogenic composition of claim 3, which is a 25-valent pneumococcal conjugate composition.

5. - the Streptococcus pneumoniae serotype 15A glycoconjugate comprises a serotype 15A capsular polysaccharide having a weight-average molecular weight (Mw) of between 75 kDa and 250 kDa, and / or - the Streptococcus pneumoniae serotype 15A glycoconjugate has a weight average molecular weight (Mw) of between 1,000 kDa and 6,000 kDa, and / or - the serotype 15A polysaccharide to carrier protein ratio (w / w) in said Streptococcus pneumoniae serotype 15A glycoconjugate is between 0.5 and 1.5, and / or - the degree of conjugation of said Streptococcus pneumoniae serotype 15A glycoconjugate is between 5 and 10, and / or - the Streptococcus pneumoniae serotype 15A glycoconjugate comprises less than about 25% free serotype 15A polysaccharide compared to the total amount of serotype 15A polysaccharide, and / or - between 50% and 90% of said serotype 15A glycoconjugates have a Kd of 0.3 or less in a CL-4B column; An immunogenic composition according to any one of claims 1 to 4.

6. The carrier protein of the Streptococcus pneumoniae serotype 15A glycoconjugate is a CRM. １９７ The immunogenic composition of any one of claims 1 to 5, wherein

7. 7. The immunogenic composition of claim 1, wherein the Streptococcus pneumoniae serotype 15A glycoconjugate is prepared using reductive amination chemistry.

8. - the Streptococcus pneumoniae serotype 23A glycoconjugate comprises a serotype 23A capsular polysaccharide having a weight-average molecular weight (Mw) of between 110 kDa and 250 kDa, and / or - the Streptococcus pneumoniae serotype 23A glycoconjugate has a weight average molecular weight (Mw) of between 1,000 kDa and 7,500 kDa, and / or - the serotype 23A polysaccharide to carrier protein ratio (w / w) in said Streptococcus pneumoniae serotype 23A glycoconjugate is between 0.5 and 1.7, and / or - the degree of conjugation of said Streptococcus pneumoniae serotype 23A glycoconjugate is between 5 and 15, and / or - the Streptococcus pneumoniae serotype 23A glycoconjugate comprises less than about 25% free serotype 23A polysaccharide compared to the total amount of serotype 23A polysaccharide; and / or Between 50% and 90% of the serotype 23A glycoconjugates have a Kd of 0.3 or less in a CL-4B column; and / or - the Streptococcus pneumoniae serotype 23A glycoconjugate comprises Streptococcus pneumoniae serotype 23A capsular polysaccharide, the Streptococcus pneumoniae serotype 23A capsular polysaccharide having a branched rhamnose content of greater than 95% compared to native Streptococcus pneumoniae serotype 23A capsular polysaccharide, which is believed to have a branched rhamnose content of about 100%; An immunogenic composition according to any one of claims 1 to 7.

9. The carrier protein of the Streptococcus pneumoniae serotype 23A glycoconjugate is a CRM. １９７ The immunogenic composition of any one of claims 1 to 8, wherein

10. 11. The immunogenic composition of any one of claims 1 to 10, wherein the Streptococcus pneumoniae serotype 23A glycoconjugate is prepared using reductive amination chemistry.

11. - the Streptococcus pneumoniae serotype 23B glycoconjugate comprises a serotype 23B capsular polysaccharide having a weight average molecular weight (Mw) of between 75 kDa and 250 kDa; - the Streptococcus pneumoniae serotype 23B glycoconjugate has a weight average molecular weight (Mw) of between 500 kDa and 4,000 kDa, and / or - the serotype 23B polysaccharide to carrier protein ratio (w / w) in the Streptococcus pneumoniae serotype 23B glycoconjugate is between 0.5 and 1.5, and / or the degree of conjugation of the Streptococcus pneumoniae serotype 23B glycoconjugate is between 5 and 12; and / or - the Streptococcus pneumoniae serotype 23B glycoconjugate comprises less than about 25% free serotype 23B polysaccharide compared to the total amount of the serotype 23B polysaccharide; and / or Between 40% and 60% of the serotype 23B glycoconjugates have a Kd of 0.3 or less in a CL-4B column. An immunogenic composition according to any one of claims 1 to 11.

12. The carrier protein of the Streptococcus pneumoniae serotype 23B glycoconjugate is a CRM. １９７ The immunogenic composition of any one of claims 1 to 11, wherein

13. 13. The immunogenic composition of any one of claims 1 to 12, wherein the Streptococcus pneumoniae serotype 23B glycoconjugate is prepared using reductive amination chemistry.

14. - the Streptococcus pneumoniae serotype 24F glycoconjugate comprises a serotype 24F capsular polysaccharide having a weight-average molecular weight (Mw) of between 120 kDa and 250 kDa, and / or - the Streptococcus pneumoniae serotype 24F glycoconjugate has a weight average molecular weight (Mw) of between 1,500 kDa and 7,500 kDa, and / or - the serotype 24F polysaccharide to carrier protein ratio (w / w) in said Streptococcus pneumoniae serotype 24F glycoconjugate is between 0.7 and 1.5, and / or - the degree of conjugation of said Streptococcus pneumoniae serotype 24F glycoconjugate is between 5 and 12, and / or - the Streptococcus pneumoniae serotype 24F glycoconjugate comprises less than about 25% free serotype 24F polysaccharide compared to the total amount of the serotype 24F polysaccharide; and / or - between 50% and 90% of said serotype 24F glycoconjugates have a Kd of 0.3 or less in a CL-4B column; and / or - the Streptococcus pneumoniae serotype 24F glycoconjugate comprises Streptococcus pneumoniae serotype 24F capsular polysaccharide, the Streptococcus pneumoniae serotype 24F capsular polysaccharide having a ribose content of greater than 95% compared to native Streptococcus pneumoniae serotype 24F capsular polysaccharide, which is believed to have a ribose content of about 100%; An immunogenic composition according to any one of claims 1 to 13.

15. The carrier protein of the Streptococcus pneumoniae serotype 24F glycoconjugate is a CRM １９７ The immunogenic composition of any one of claims 1 to 14, wherein

16. 16. The immunogenic composition of any one of claims 1 to 15, wherein the Streptococcus pneumoniae serotype 24F glycoconjugate is prepared using reductive amination chemistry.

17. - the Streptococcus pneumoniae serotype 35B glycoconjugate comprises a serotype 35B capsular polysaccharide having a weight average molecular weight (Mw) of between 25 kDa and 50 kDa, and / or - the Streptococcus pneumoniae serotype 35B glycoconjugate has a weight average molecular weight (Mw) of between 500 kDa and 5,000 kDa, and / or - the serotype 35B polysaccharide to carrier protein ratio (w / w) in said Streptococcus pneumoniae serotype 35B glycoconjugate is between 0.4 and 2.0, and / or - the degree of conjugation of said Streptococcus pneumoniae serotype 35B glycoconjugate is between 5 and 10, and / or - the Streptococcus pneumoniae serotype 35B glycoconjugate comprises less than about 20% free serotype 35B polysaccharide compared to the total amount of said serotype 35B polysaccharide, and / or - between 50% and 80% of said serotype 35B glycoconjugates have a Kd of 0.3 or less in a CL-4B column; 17. The immunogenic composition of any one of claims 1 to 16.

18. The carrier protein of the Streptococcus pneumoniae serotype 35B glycoconjugate is a CRM. １９７ 18. The immunogenic composition of any one of claims 1 to 17, wherein

19. 19. The immunogenic composition of any one of claims 1 to 18, wherein the Streptococcus pneumoniae serotype 35B glycoconjugate is prepared using reductive amination chemistry.

20. 20. The immunogenic composition of any one of claims 1 to 19, wherein all of the glycoconjugates are individually conjugated to the carrier protein.

21. Glycoconjugates derived from Streptococcus pneumoniae serotype 3 are used in CRMs. １９７ 21. The immunogenic composition of any one of claims 1 to 20, wherein the immunogenic composition is conjugated to

22. 21. The immunogenic composition of any one of claims 1 to 20, wherein the glycoconjugate derived from Streptococcus pneumoniae serotype 3 is conjugated to an SCP.

23. The glycoconjugates derived from Streptococcus pneumoniae serotypes 8, 10A, 11A, 12F, 15B, 22F, and 33F are CRMs. １９７ 23. The immunogenic composition of any one of claims 1 to 22, wherein the immunogenic composition is conjugated to

24. The glycoconjugates derived from Streptococcus pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F are CRMs. １９７ 24. The immunogenic composition of any one of claims 1 to 23, wherein the immunogenic composition is conjugated to

25. The glycoconjugate is a CRM １９７ 22. The immunogenic composition of claim 1, wherein all of the IgG1-specific antigens are individually conjugated to:

26. Glycoconjugates derived from Streptococcus pneumoniae serotype 3 are conjugated to SCPs, and other glycoconjugates are conjugated to CRMs. １９７ 21. The immunogenic composition of claim 1, wherein all of the IgG1-specific antibodies are individually conjugated to:

27. The glycoconjugate derived from Streptococcus pneumoniae serotype 3 comprises a serotype 3 saccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII): 【Chemistry 1】 wherein X is CH ２ (CH ２ ) ｎ’ , (CH ２ CH ２ O) ｍ CH ２ CH ２ , NHCO(CH ２ ) ｎ’ , NHCO(CH ２ CH ２ O) ｍ CH ２ CH ２ , OCH ２ (CH ２ ) ｎ’ and O(CH ２ CH ２ O) ｍ CH ２ CH ２ wherein n′ is selected from 1 to 10 and m is selected from 1 to 4; X' is CH ２ O (CH ２ ) ｎ’’ CH ２ C=O, CH ２ O (CH ２ CH ２ O) ｍ’ (CH ２ ) ｎ’’ CH ２ C═O, n″ is selected from 0 to 10, and m′ is selected from 0 to 4.

27. The immunogenic composition of any one of claims 1 to 26, having the following structure:

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