Adjuvanted immunogenic compositions comprising conjugated pneumococcal capsular saccharide antigens and uses thereof

Abstract

The present invention relates to new adjuvanted immunogenic compositions comprising conjugated S. pneumoniae capsular saccharide antigens (saccharide conjugates) and liposomal adjuvants containing saponins. The immunogenic compositions of the invention generally comprise saccharide conjugates wherein the saccharide is from a serotype of S. pneumoniae. The invention also relates to the use of the new immunogenic compositions for the vaccination of human individuals, in particular infants and the elderly, against S. pneumoniae infection.

Description

Technical Field

[0001] This invention relates to novel adjuvanted immunogenic compositions comprising a conjugated Streptococcus pneumoniae capsular glycoantigen (glycoconjugate) and a saponin-containing liposomal adjuvant. The immunogenic compositions of this invention typically comprise a glycoconjugate, wherein the sugar is derived from Streptococcus pneumoniae (…). Streptococcus pneumoniae The invention also relates to the vaccination of human individuals (particularly infants and the elderly) with the use of this novel immunogenic composition to combat Streptococcus pneumoniae infection. 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 manifestations of invasive Streptococcus pneumoniae infections, while the spread of the bacteria in the respiratory tract can lead to middle ear infections, sinusitis, or recurrent bronchitis. Non-invasive manifestations are generally less severe than invasive manifestations, but are significantly more common.

[0003] In Europe and the United States, pneumococcal pneumonia is the most common community-acquired bacterial pneumonia, with an estimated 100 infections per 100,000 adults annually. The corresponding figures for febrile bacteremia and meningitis are 15–19 per 100,000 and 1–2 per 100,000, respectively. Infants and the elderly, as well as immunocompromised individuals of any age, are at significantly higher risk of developing one or more of these manifestations. Even in economically developed regions, the mortality rate of invasive pneumococcal disease is high; the average mortality rate for adult pneumococcal pneumonia is 10%–20%, while the mortality rate in high-risk groups can exceed 50%. To date, pneumonia is the leading cause of death from pneumococcal pneumonia worldwide.

[0004] The causative agent of pneumococcal disease, Streptococcus pneumoniae (pneumococcus), is a Gram-positive cyst coccus surrounded by a polysaccharide capsule. Differences in the composition of this capsule allow for serological differentiation among approximately 91 capsule types, some of which are commonly associated with pneumococcal disease, while others are almost entirely unrelated. Invasive pneumococcal infections include pneumonia, meningitis, and febrile bacteremia; common non-invasive manifestations include otitis media, sinusitis, and bronchitis.

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

[0006] The recent development of widespread resistance to essential antibiotics among microorganisms and the increasing number of immunocompromised individuals underscore the need for pneumococcal vaccines with broader protective effects.

[0007] Specifically, it is necessary to address the unmet coverage issues caused by not being included in Prevnar13. ® The medical need for pneumococcal disease caused by serotypes identified in the study and potential serotype replacements over time. (Except for Prevnar 13) ® The pathogenicity-specific serotypes beyond the 13 listed in the study vary by region and population, and may change over time due to long-term trends in acquired antibiotic resistance, introduction of pneumococcal vaccines, and unknown sources. There is a need for immunogenic compositions that can be used to induce an immune response against additional pneumococcal serotypes in humans, particularly in children under 2 years of age.

[0008] The purpose of the novel immunogenic compositions of the present invention is to provide resistance against antibodies not present in Prevnar 20. TM Appropriate protection against additional Streptococcus pneumoniae serotypes found in Vaxneuvance. In one aspect, the immunogenic compositions of the present invention aim to provide protection against those not found in Vaxneuvance. TM and / or Prevnar 20 TM The vaccine provides appropriate protection against additional pneumococcal serotypes found in the vaccine, while maintaining an immune response against serotypes currently covered by the vaccine. Attached Figure Description

[0009] Figure 1 : The effect of QS-21-containing liposome adjuvant on the OPA titer of a quarter-dose of PCV20. ST3-specific opsonization titer was measured in serum collected from juvenile rhesus macaques 4 weeks after the first dose. Each point represents an individual animal and data are expressed as the geometric mean titer of OPA with a 95% confidence interval. LLOQ: Lower limit of quantitation. QS-21 adj: Liposome adjuvant containing QS-21.

[0010] Figure 2: Effect of QS-21-containing liposome adjuvant on the titer of a quarter-dose of PCV20 IgG. ST3-specific IgG titer was measured in serum collected from juvenile rhesus monkeys 4 weeks after the first dose. Each point represents an individual animal and data are expressed in µg / ml of IgG with a 95% confidence interval. LLOQ: Limit of quantitation. QS-21 adj: Liposome adjuvant containing QS-21. Summary of the Invention

[0011] 1. The pneumococcal glycoconjugate of the present invention This invention relates in part to conjugated Streptococcus pneumoniae capsular sugar antigens (also known as glycoconjugates). For the purposes of this invention, the term "glycoconjugate" refers to a Streptococcus pneumoniae capsular sugar conjugated to a carrier protein via covalent or non-covalent bonds. In one embodiment, the Streptococcus pneumoniae capsular sugar is conjugated to a carrier protein (such as the rhizavidin / biotin system, see, for example, WO2012155007, WO2020056202) via non-covalent bonds. The capsular sugar is preferably conjugated via covalent bonds. In one embodiment, the capsular sugar is directly conjugated to the carrier protein. In a second embodiment, the capsular sugar is conjugated to the carrier protein via a spacer / linker.

[0012] 1.1 The pneumococcal capsular sugar of the present invention Throughout this specification, the term "sugar" may refer to a polysaccharide or an oligosaccharide and includes both. In one embodiment, the sugar of the present invention may be an oligosaccharide. Oligosaccharides have a small number of repeating units (typically 5 to 15 repeating units) and are typically produced synthetically or by hydrolysis of polysaccharides. However, all sugars of the present invention and in the immunogenic compositions thereof are preferably polysaccharides. High molecular weight polysaccharides can induce specific antibody immune responses due to epitopes present on the surface of antigens. Preferably, isolated and purified high molecular weight capsular polysaccharides are considered for use in the conjugates, compositions, and methods of the present invention. Therefore, in a preferred embodiment of the present invention, the sugar is a polysaccharide.

[0013] The sugar used in this invention is preferably pneumococcal capsular sugar (also referred to herein as "pneumococcal capsular sugar"). S. pneumoniae Capsular saccharide (or "capsular sugar") is found in several medically important bacteria. Bacterial capsules are primarily composed of polysaccharides. Capsular saccharide is prepared using standard techniques known to those skilled in the art.

[0014] In one embodiment, the pneumococcal capsular sugar used in this invention is a synthetic carbohydrate.

[0015] However, in a preferred embodiment, the source of the Streptococcus pneumoniae capsular sugar according to the invention can be Streptococcus pneumoniae cells. Streptococcus pneumoniae strains that can be used as a source of capsular sugar can be obtained from established culture collections (e.g., from the United States Type Culture Collection (ATCC, Manassas, VA USA) or a Streptococcus Reference Laboratory (Centers for Disease Control and Prevention, Atlanta, GA USA)) or clinical samples.

[0016] Streptococcus pneumoniae capsular sugar can be obtained directly from the bacteria using isolation methods known to those skilled in the art (see, for example, the methods disclosed in US2006 / 0228380, US2006 / 0228381, US2007 / 0184071, US2007 / 0184072, US2007 / 0231340, US2008 / 0102498, and WO2008 / 118752). Streptococcus pneumoniae capsular sugar can also be produced using synthetic methods known to those skilled in the art.

[0017] If the Streptococcus pneumoniae capsular sugar is obtained directly from the bacteria, the bacterial cells can be grown in a culture medium (e.g., in a soybean-based medium). After fermentation, the bacterial cells can be lysed to produce cell lysates. The capsular sugar can then be separated from the cell lysates using purification techniques known in the art, including centrifugation, deep filtration, precipitation, ultrafiltration, treatment with activated carbon, percolation, and / or column chromatography (see, for example, US2006 / 0228380, US2006 / 0228381, and WO2008 / 118752). Subsequently, the purified capsular sugar can be used to prepare immunogenic conjugates.

[0018] The isolated capsular sugars obtained through purification can be characterized by various parameters, including, for example, weight-average molecular weight (Mw). The molecular weight of polysaccharides can be measured by a combination of size exclusion chromatography (SEC) and multi-angle laser scattering detectors (MALLS).

[0019] The capsular sugar used in this invention is a capsular sugar derived from Streptococcus pneumoniae.

[0020] The capsular sugar used in this invention is preferably a capsular sugar selected from the following Streptococcus pneumoniae serotypes: serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9V, 9N, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11E, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18 B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A , 32F, 33A, 33B, 33C, 33D, 33E, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F and 48.

[0021] The capsular sugar used in this invention is preferably a capsular sugar selected from the following Streptococcus pneumoniae serotypes: serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73.

[0022] Streptococcus pneumoniae capsular polysaccharide comprises repeating oligosaccharide units, each of which may contain up to 8 sugar residues.

[0023] In one embodiment, the capsular sugar of the present invention may be an oligosaccharide unit, or a sugar chain shorter than the natural length of a repeating oligosaccharide unit. In one embodiment, the capsular sugar of the present invention is a repeating oligosaccharide unit of a relevant serotype.

[0024] In one embodiment, the capsular sugar of the present invention may be an oligosaccharide. Oligosaccharides have a small number of repeating units (typically 5 to 15 repeating units) and are typically produced synthetically or through polysaccharide hydrolysis.

[0025] In a preferred embodiment, the capsular sugar of the present invention is a polysaccharide. Due to the epitopes present on the antigen surface, high molecular weight capsular polysaccharides can induce specific antibody immune responses. Preferably, isolated and purified high molecular weight capsular polysaccharides are considered for use in the conjugates, compositions, and methods of the present invention.

[0026] In a preferred embodiment, the isolated capsular sugar (purified prior to further processing) used in this invention has a weight-average molecular weight between 50 kDa and 5000 kDa. In a preferred embodiment, the capsular sugar used in this invention has a weight-average molecular weight between 200 kDa and 4000 kDa.

[0027] Consider any integer within any of the above ranges as an embodiment of this disclosure.

[0028] 1.2 Characteristics of the pneumococcal glycoconjugate of the present invention Generally, conjugation of sugars to carriers enhances the immunogenicity of sugars because it converts sugars from T-independent antigens to T-dependent antigens, thus allowing the initiation of immune memory. Conjugation is particularly useful for pediatric vaccines.

[0029] The isolated capsular sugars can be activated (e.g., chemically activated) to enable them to react (e.g., with the linker or directly with the carrier protein) and subsequently incorporated into the glycoconjugate, as further described herein.

[0030] Prior to activation, the size of the isolated polysaccharide can be reduced while retaining key structural features. Size setting may be performed using mechanical or chemical methods. In one embodiment, the size of the isolated polysaccharide is reduced by chemical hydrolysis. Chemical hydrolysis may be carried out using a weak acid (e.g., acetic acid, formic acid, propionic acid). In one embodiment, chemical hydrolysis is carried out using formic acid. In another embodiment, chemical hydrolysis is carried out using propionic acid. In a preferred embodiment, chemical hydrolysis is carried out using acetic acid. Chemical hydrolysis may also be carried out using diluted strong acids (such as diluted hydrochloric acid, diluted sulfuric acid, diluted phosphoric acid, diluted nitric acid, or diluted perchloric acid). In one embodiment, chemical hydrolysis is carried out using diluted hydrochloric acid. In another embodiment, chemical hydrolysis is carried out using diluted sulfuric acid. In another embodiment, chemical hydrolysis is carried out using diluted phosphoric acid. In another embodiment, chemical hydrolysis is carried out using diluted nitric acid. In another embodiment, chemical hydrolysis is carried out using diluted perchloric acid.

[0031] The size of the isolated polysaccharides can also be reduced by mechanical homogenization. In one embodiment, the size of the isolated polysaccharides is reduced by high-pressure homogenization. High-pressure homogenization achieves a high shear rate by pumping the process flow through a sufficiently small flow path. The shear rate is increased by using a larger applied homogenization pressure, and the exposure time can be increased by recirculating the feed flow through the homogenizer.

[0032] The weight-average molecular weight (Mw) of a sugar prior to conjugation refers to the Mw before sugar activation (i.e., after the final size setting step but before the sugar reacts with the activator). In the context of this invention, the Mw of the sugar is substantially unmodified by the activation step, and the Mw of the sugar incorporated into the conjugate is similar to the Mw of the sugar measured prior to activation.

[0033] In one embodiment, the pneumococcal glycoconjugate of the present invention comprises pneumococcal capsular sugar, wherein the weight-average molecular weight (Mw) of the sugar prior to conjugation is between 50 kDa and 1,000 kDa.

[0034] In one embodiment, the pneumococcal glycoconjugate of the present invention comprises pneumococcal capsular sugar, wherein the weight-average molecular weight (Mw) of the sugar prior to conjugation is between 50 kDa and 600 kDa.

[0035] In one embodiment, the pneumococcal glycoconjugate of the present invention comprises pneumococcal capsular sugar, wherein the weight-average molecular weight (Mw) of the sugar prior to conjugation is between 50 kDa and 500 kDa.

[0036] In some embodiments, the pneumococcal glycoconjugate of the present invention has a weight-average molecular weight (Mw) between 250 kDa and 20,000 kDa.

[0037] In some embodiments, the pneumococcal glycoconjugate of the present invention has a weight-average molecular weight (Mw) between 500 kDa and 15,000 kDa.

[0038] Another way to characterize the pneumococcal glycoconjugates of the present invention is through a carrier protein (e.g., CRM) that is conjugated with the glycoconjugate. 197 The number of lysine residues in the polysaccharide (SCP, DT, or TT) can be used to characterize the sugar by the range of conjugated lysine residues (degree of conjugation). Evidence of lysine modification of the carrier protein due to covalent linkage with the polysaccharide can be obtained by amino acid analysis using conventional methods known to those skilled in the art. Conjugation can result in a reduced number of recovered lysine residues compared to the carrier protein starting material used to generate the conjugated material. In a preferred embodiment, the degree of conjugation of the pneumococcal glycoconjugate of the present invention is between 2 and 15.

[0039] The Streptococcus pneumoniae glycoconjugates of the present invention can also be characterized by a sugar to carrier protein ratio (w / w). In some embodiments, the w / w ratio of Streptococcus pneumoniae capsular sugar to carrier protein in the glycoconjugate is between 0.5 and 3.0. In other embodiments, the w / w ratio is between 0.5 and 2.0. In other embodiments, the w / w ratio is between 0.5 and 1.5. In other embodiments, the w / w ratio is between 0.8 and 1.2. In other embodiments, the w / w ratio is between 0.5 and 1.0. In other embodiments, the w / w ratio is between 1.0 and 1.5. In other embodiments, the w / w ratio is between 1.0 and 2.0. In a further embodiment, the w / w ratio is between 0.8 and 1.2.

[0040] The pneumococcal glycoconjugates and immunogenic compositions of the present invention may contain free sugars that are not conjugated to the carrier protein but are still present in the glycoconjugate composition. The free sugars may non-covalently associate with the glycoconjugates (i.e., non-covalently bind to the glycoconjugates, adsorb onto the glycoconjugates, or be embedded in or encapsulated by the glycoconjugates). In a preferred embodiment, each pneumococcal glycoconjugate of the present invention contains less than about 50% free pneumococcal sugar compared to the total sugar content. In a preferred embodiment, each pneumococcal glycoconjugate of the present invention contains less than about 25% free pneumococcal sugar compared to the total sugar content.

[0041] Streptococcus pneumoniae glycoconjugates can also be analyzed using their molecular size distribution (K). d Characterization. Size exclusion chromatography medium (CL-4B) can be used to determine the relative molecular size distribution of conjugates. Size exclusion chromatography (SEC) is used in gravity-feed columns to obtain the molecular size distribution profile of conjugates. Large molecules excluded from the pores of the medium elute faster than small molecules. The column eluent is collected using a fraction collector. The fraction is determined colorimetrically by a sugar assay. To determine K... d The column was calibrated to confirm that the molecule was a completely size-restricted fraction (V0), (K d =0), and the flow fraction representing the maximum retention (V) i ), (K d =1). The fraction (V) that achieves specific sample properties. e ) and K d The relation is K d =(V e -V0) / (V i -V0).

[0042] In a preferred embodiment, at least 30% of the pneumococcal glycoconjugates of the present invention have a K0.3 or less in a CL-4B column. d In a preferred embodiment, at least 40% of the glycoconjugates have a K0.3 or less in the CL-4B column. d In a preferred embodiment, the pneumococcal glycoconjugate of the present invention, at concentrations between 40% and 80%, has a K0.3 or greater in a CL-4B column. d .

[0043] 1.3 Preparation method of the pneumococcal glycoconjugate of the present invention The pneumococcal glycoconjugate of the present invention can be prepared using any coupling technique known to those skilled in the art.

[0044] In one embodiment, the pneumococcal capsular sugar is coupled to a carrier protein via a non-covalent bond (see, for example, WO2012155007, WO2020056202).

[0045] In one embodiment, the pneumococcal capsular sugar is covalently conjugated. In another embodiment, the capsular sugar is directly conjugated to the carrier protein. In a second embodiment, the capsular sugar is conjugated to the carrier protein via a spacer / linker.

[0046] In one embodiment, some or all of the pneumococcal glycoconjugates of the present invention are bound to a carrier protein via a linker (e.g., a bifunctional linker). The linker is optionally heterobifunctional or homobifunctional, having, for example, a reactive amino group and a reactive carboxylic acid group, two reactive amino groups, or two reactive carboxylic acid groups. The linker has, for example, between 4 and 20, 4 and 12, or 5 and 10 carbon atoms.

[0047] Possible linkers include adipic dihydrazide (ADH). Other linkers include β-propionamide (WO 00 / 10599), nitrophenylethylamine (Gever et al. (1979) Med. Microbiol. lmmunol. 165; 171-288), haloalkyl halides (US4057685), glycosidic bonds (US4673574, US4808700), hexamethylenediamine, and 6-aminohexanoic acid (US4459286).

[0048] In one embodiment, some or all of the pneumococcal glycoconjugates of the present invention are directly conjugated to a carrier protein (without a linker).

[0049] Generally, the following types of chemical groups on protein carriers can be used for coupling / conjugation: 1) Amino group (e.g., via lysine). In one embodiment, this group is directly attached to a carboxyl group on the sugar or chemically attached to a carboxyl group on a linker via a carbodiimide, for example using EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide). In another embodiment, this group is directly attached to a hydroxyl group on the sugar activated by CDAP or CNBr or attached to such a group on a linker; attached to a sugar or a linker having an aldehyde group; attached to a sugar or a linker having a succinimide ester group.

[0050] 2) Carboxyl group (e.g., via aspartic acid or glutamic acid). In one embodiment, this group is directly attached to the amino group on the sugar or chemically attached to the amino group on the linker via a carbodiimide, for example, using EDAC.

[0051] 3) Thiol group (e.g., via cysteine). In one embodiment, this group is attached to a bromo- or chloroacetylated sugar or chemically attached to a connector via maleimide. In one embodiment, this group is activated / modified with diazidobenzidine.

[0052] 4) Hydroxyl group (e.g., via tyrosine). In one embodiment, this group is activated / modified with diazidobenzidine.

[0053] 5) Imidazole group (e.g., via histidine). In one embodiment, this group is activated / modified with diazinonidine.

[0054] 6) Guanidin (e.g., via arginine).

[0055] 7) Indole group (e.g., via tryptophan).

[0056] On pneumococcal sugars, the following groups are typically available for coupling: OH, COOH, or NH2. Aldehyde groups can be generated following various treatments known in the art, such as periodate, acid hydrolysis, and hydrogen peroxide.

[0057] In one embodiment, some or all of the pneumococcal glycoconjugates of the present invention are prepared using CDAP chemistry. In this embodiment, the pneumococcal sugar is activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. Subsequently, the activated sugar can be coupled directly or via a spacer (linker) group to an amino group on a carrier protein. For example, the spacer may be cystamine or cysteamine that produces a thiolated polysaccharide that can be coupled to the carrier via a thioether bond obtained after reacting with a maleimide-activated carrier protein (e.g., using N-[γ-maleiminobutyryloxy]succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g., using iodoacetimide, bromoacetate N-succinimide ester (SBA; SIB), (4-iodoacetyl)aminobenzoic acid N-succinimide ester (SlAB), (4-iodoacetyl)aminobenzoic acid sulfosuccinimide ester (sulfo-SIAB), iodoacetate N-succinimide ester (SIA), or 3-[bromoacetamido]propionic acid succinimide ester (SBAP)).

[0058] In a preferred embodiment, the cyanate ester of the activated sugar is coupled with hexamethylenediamine or adipic hydrazide (ADH), and the amino-derived sugar is conjugated to the carrier protein via a carbodiimide (e.g., EDAC or EDC) chemistry through the carboxyl group on the protein carrier. Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348 and WO 96 / 129094.

[0059] In one embodiment, some or all of the pneumococcal glycoconjugates of the present invention are prepared using carbodiimide, hydrazide, active ester, norcamphene, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many such techniques are described in International Patent Application Publication No. WO 98 / 42721. Conjugation may involve a carbonyl linker, which can be formed by reacting the free hydroxyl group of the sugar with CDI (see Bethell et al. (1979) 1. Biol. Chern. 254: 2572-2574; Hearn et al. (1981) J. Chromatogr. 218: 509-518), followed by reaction with a protein to form a carbamate bond. This may involve reducing the 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.

[0060] direct reductive amination In one embodiment, some or all of the pneumococcal glycoconjugates of the present invention are prepared by direct reductive amination (see, for example, US 4365170, US 4673574, WO2006 / 110381, WO2008 / 079653, WO2008 / 143709, WO2008 / 079732, WO2011 / 110531, WO2012 / 119972, WO2015110941, WO2015110940, WO2018 / 144439, WO2018 / 156491).

[0061] According to the present invention, reductive amination comprises two steps: (1) oxidizing (activating) the purified Streptococcus pneumoniae sugar, and (2) reacting the activated sugar with a carrier protein (e.g., CRM). 197 (TT or SCP) reduction to form glycoconjugates.

[0062] As described above, the pneumococcal sugar can be sized to a target molecular weight (MW) range prior to oxidation. Therefore, in one embodiment, the isolated polysaccharide is sized prior to oxidation.

[0063] In one embodiment, the pneumococcal sugar of the present invention is conjugated to a carrier protein via a method comprising the following steps: (a) Reacting the pneumococcal sugar with an oxidizing agent; (b) Mix the activated sugar from step (a) with the carrier protein; and (c) React the mixed activated sugar and carrier protein with a reducing agent to form a glycoconjugate.

[0064] In one embodiment, the pneumococcal sugar of the present invention is conjugated to a carrier protein via a method comprising the following steps: (a) Reacting the pneumococcal sugar with an oxidizing agent; (a') Quenching the oxidation reaction by adding a quenching agent; (b) Mix the activated sugar from step (a') with the carrier protein; and (c) React the mixed activated sugar and carrier protein with a reducing agent to form a glycoconjugate.

[0065] After oxidation step (a), the sugar is activated and referred to as "activated sugar".

[0066] In one embodiment, the oxidant is any oxidant that oxidizes a terminal hydroxyl group to an aldehyde. In one embodiment, the oxidant is periodate. For the purposes of this invention, the term "periodate" includes periodate and periodic acid; the term also includes metaperiodate (IO4). -) and periodate (IO6) 5- And various salts of periodic acid (such as sodium periodate and potassium periodate).

[0067] In one embodiment, the oxidant is periodate in the presence of a divalent cation (see WO2008 / 143709).

[0068] 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 in Mg... 2+ The presence of periodic acid is described. In one embodiment, the oxidant is in Ca... 2+ The presence of periodic acid is a given. In one embodiment, the oxidant is periodate.

[0069] In a preferred embodiment, the oxidant is sodium periodate. In one embodiment, the periodate used for oxidation is metaperiodate. In another embodiment, the periodate used for oxidation is sodium metaperiodate.

[0070] When polysaccharides react with periodate, periodate oxidizes the ortho-hydroxyl group to form a carbonyl or aldehyde group, and causes C / C bond cleavage. For this reason, the term "reacting polysaccharides with periodate" includes the oxidation of the ortho-hydroxyl group by periodate.

[0071] In one embodiment, step a) comprises reacting the polysaccharide with 0.01 to 2 molar equivalents of periodate. In one embodiment, step a) comprises reacting the polysaccharide with 0.1 to 1.0 molar equivalents of periodate. In one embodiment, step a) comprises reacting the polysaccharide with 0.1 to 0.5 molar equivalents of periodate.

[0072] In one embodiment, the oxidant is a mixture of a stable nitryl radical compound and an oxidant (see WO2014097099).

[0073] In one aspect, the stable nitroglycerin radical compound is a molecule containing a TEMPO or PROXYL (2,2,5,5-tetramethyl-1-pyrrolyloxy) moiety. Preferably, the molecule is capable of selectively oxidizing a primary alcohol in the presence of an oxidant to produce an aldehyde group without affecting the secondary hydroxyl group. More preferably, the molecule is capable of selectively oxidizing a primary alcohol in the presence of an oxidant to produce an aldehyde group without over-oxidizing it to a carboxyl group. In one aspect, the stable nitryl radical compound is TEMPO, 2,2,6,6-tetramethyl-4-(methanesulfonyloxy)-1-piperidinyloxy, 4-phosphono-TEMPO, 4-oxo-TEMPO, 4-methoxy-TEMPO, 4-isothiocyano-TEMPO, 4-(2-iodoacetamido)-TEMPO radical, 4-hydroxy-TEMPO, 4-cyano-TEMPO, 4-carboxyl-TEMPO, 4-(2-bromoacetamido)-TEMPO, or 4-amino-TEMPO, 4-acetamido-2,2,6,6-tetramethylpiperidin-1-oxy. Preferably, the stable nitryl radical compound is TEMPO. In one aspect, the stable nitryl radical compound is selected from the group consisting of: TEMPO, 2,2,6,6-tetramethyl-4-(methanesulfonyloxy)-1-piperidinyloxy, 4-phosphono-TEMPO, 4-oxo-TEMPO, 4-methoxy-TEMPO, 4-isothiocyano-TEMPO, 4-(2-iodoacetamido)-TEMPO radical, 4-hydroxy-TEMPO, 4-cyano-TEMPO, 4-carboxyl-TEMPO, 4-(2-bromoacetamido)-TEMPO, 4-amino-TEMPO, and 4-acetamido-2,2,6,6-tetramethylpiperidin-1-oxy. Preferably, the stable nitryl radical compound is TEMPO. In another respect, the stable nitryl radical compound is 3β-DOXYL-5α-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid, methyl 5-DOXYL-stearate, 3-(aminomethyl)-PROXYL, 3-carbamoyl-PROXYL, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolino-1-oxy, 3-carboxyl-PROXYL, or 3-cyano-PROXYL. In another aspect, the stable nitryl radical compound is selected from the group consisting of: 3β-DOXYL-5α-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid, methyl 5-DOXYL-stearate, 3-(aminomethyl)-PROXYL, 3-carbamoyl-PROXYL, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolino-1-oxy, 3-carboxy-PROXYL, and 3-cyano-PROXYL.

[0074] In one aspect, the oxidant is a molecule with an N-halogenated moiety. Preferably, the molecule is capable of selectively oxidizing primary alcohols in the presence of a nitrocellulose radical compound. In one aspect, the oxidant is N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid, or 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. In one aspect, the oxidizing agent is selected from the group consisting of: N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid, and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. Preferably, the oxidizing agent is N-chlorosuccinimide.

[0075] In one aspect, the stable nitryl radical compound is 2,2,6,6-tetramethyl-1-piperidinyloxy radical (TEMPO), and the oxidant is N-chlorosuccinimide (NCS).

[0076] In one embodiment, the quencher in step a') is selected from vicinal diols, 1,2-amino alcohols, amino acids, glutathione, sulfites, bisulfites, dithionites, metabisulfites, thiosulfates, phosphites, hypophosphites, or phosphorous acid.

[0077] In one embodiment, the quencher is a 1,2-amino alcohol of formula (I): Where R 1 Selected from H, methyl, ethyl, propyl or isopropyl.

[0078] In one embodiment, the quenching agent is selected from sodium sulfite and potassium salt, sodium bisulfite and potassium salt, sodium dithionite and potassium salt, sodium metabisulfite and potassium salt, sodium thiosulfate and potassium salt, sodium phosphite and potassium salt, sodium hypophosphite and potassium salt, or sodium phosphite and potassium salt.

[0079] In one embodiment, the quencher is an amino acid. In such embodiments, the amino acid may be selected from serine, threonine, cysteine, cystine, methionine, proline, hydroxyproline, tryptophan, tyrosine, and histidine.

[0080] In one embodiment, the quenching agent is a sulfite, such as bisulfite, dithionite, metabisulfite, or thiosulfate.

[0081] In one embodiment, the quencher is a compound containing two ortho-hydroxyl groups (ortho-diols), i.e., the two hydroxyl groups are covalently linked to two adjacent carbon atoms.

[0082] The preferred quenching agent is a compound of formula (II): Where R 1 and R 2 Each is independently selected from H, methyl, ethyl, propyl or isopropyl.

[0083] In a preferred embodiment, the quencher is glycerol, ethylene glycol, propylene-1,2-diol, butane-1,2-diol, or butane-2,3-diol or ascorbic acid. In an even more preferred embodiment, the quencher is butane-2,3-diol.

[0084] In a preferred embodiment, the degree of oxidation of the activated pneumococcal sugar (also referred to herein as "degree of activation") is between 2 and 30. In one embodiment, the degree of oxidation (DO) of the activated pneumococcal sugar is between 10 and 25.

[0085] In one embodiment, the activated sugar and carrier protein are freeze-dried prior to step b).

[0086] In one embodiment, the initial input ratio (by weight) of the activated pneumococcal sugar to the carrier protein in step b) is between 4:1 and 0.1:1. In another embodiment, the initial input ratio (by weight) of the activated pneumococcal sugar to the carrier protein in step b) is between 1.5:1 and 0.5:1.

[0087] In one embodiment, the reduction reaction (c) is carried out in an aqueous solvent. In another embodiment, the reduction reaction (c) is carried out in an aprotic solvent.

[0088] In one embodiment, the reduction reaction (c) is carried out in the presence of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). In one embodiment, the reduction reaction (c) is carried out in the presence of dimethylformamide (DMF). In one embodiment, the reduction reaction (c) is carried out in the presence of dimethyl sulfoxide (DMSO).

[0089] In one embodiment, the reduction reaction (c) is carried out in a solution consisting essentially of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). In another embodiment, the reduction reaction (c) is carried out in a solution consisting essentially of dimethylformamide (DMF). In yet another embodiment, the reduction reaction (c) is carried out in a solution consisting essentially of dimethyl sulfoxide (DMSO).

[0090] In one embodiment, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent.

[0091] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride or zinc borohydride in the presence of Bronsted acid or Lewis acid, amine boranes such as pyridineborane, 2-methylpyridineborane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe 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).

[0092] In one embodiment, a reducing agent in step c) is used in an amount between 0.2 and 20 molar equivalents. In another embodiment, a reducing agent in step c) is used in an amount between 0.5 and 10 molar equivalents. In yet another embodiment, a reducing agent in step c) is used in an amount between 1.0 and 5 molar equivalents.

[0093] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate. These aldehyde groups can be capped using a suitable capping agent. In one embodiment, the capping agent is sodium borohydride (NaBH4). In one embodiment, capping is achieved by mixing the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In one embodiment, capping is achieved by mixing the product of step c) with 1 to 10 molar equivalents of sodium borohydride. In one embodiment, capping is achieved by mixing the product of step c) with 1 to 5 molar equivalents of sodium borohydride.

[0094] CDI and / or CDT chemistry In one embodiment, some or all of the pneumococcal glycoconjugates of the present invention are prepared by CDI and / or CDT chemistry, as disclosed in WO2022249107.

[0095] CDI and / or CDT chemistry involves two steps: (1) reacting pneumococcal sugar with CDI and / or CDT in an aprotic solvent to produce activated sugar (activation), and (2) reacting the activated sugar with a carrier protein (e.g., CRM). 197 (TT or SCP) reaction to form glycoconjugates.

[0096] In one embodiment, the activator in step (1) is 1,1'-carbonyldiimidazole (CDI). In another embodiment, the activator in step (1) is 1,1'-carbonyl-bis-(1,2,4-triazole) (CDT).

[0097] As mentioned above, the size of the pneumococcal sugar can be set to the target molecular weight (MW) range before activation with CDI and / or CDT.

[0098] Therefore, in one embodiment, the pneumococcal sugar is size-set prior to activation with CDI. In one embodiment, the isolated polysaccharide is size-set prior to activation with CDT. In one embodiment, the pneumococcal sugar is size-set to any of the target molecular weight (MW) ranges defined above.

[0099] Therefore, in one embodiment, the pneumococcal sugar is conjugated to the carrier protein by a method comprising the following steps: (a) React the isolated polysaccharide with CDI and / or CDT in an aprotic solvent; (b) React the activated polysaccharide from step (a) with the carrier protein in an aprotic solvent to form a glycoconjugate.

[0100] After step (a), the polysaccharide is referred to as activated and is called "activated polysaccharide".

[0101] In one embodiment, step a) comprises reacting pneumococcal sugar with CDI.

[0102] In one embodiment, step a) comprises reacting pneumococcal sugar with an amount of CDI between 0.5 and 10 molar equivalents relative to the amount of pneumococcal sugar present in the reaction mixture.

[0103] In one implementation, step a) comprises reacting pneumococcal sugar with CDT.

[0104] In one embodiment, step a) comprises reacting pneumococcal sugar with an amount of CDT between 0.5 and 10 molar equivalents relative to the amount of pneumococcal sugar present in the reaction mixture.

[0105] In one embodiment, activation reaction a) is carried out in the presence of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide, N-methyl-2-pyrrolidone, or hexamethylphosphoramide (HMPA). In one embodiment, activation reaction a) is carried out in the presence of dimethyl sulfoxide (DMSO).

[0106] In one embodiment, the activation reaction a) is carried out in a solution consisting essentially of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF).

[0107] In one embodiment, the conjugation reaction b) is carried out in the presence of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide, N-methyl-2-pyrrolidone, or hexamethylphosphoramide (HMPA).

[0108] In one embodiment, the conjugation reaction b) is carried out in the presence of dimethyl sulfoxide (DMSO).

[0109] In one embodiment, the conjugation reaction b) is carried out in a solution consisting essentially of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF).

[0110] In one embodiment, a weak organic base may be added to the reaction mixture after activation reaction a) but before conjugation reaction b). The weak organic base may be added before or after the introduction of the carrier protein into the reaction mixture. Therefore, in one embodiment, the weak organic base is added to the reaction mixture before the introduction of the carrier protein. In another embodiment, the weak organic base is added to the reaction mixture after the introduction of the carrier protein. The weak organic base may be selected from alkylamines, imidazoles, triazoles, pyridines, histidines, and guanidines. Alkylamines include alkyl primary amines, such as methylamine, ethylamine, propylamine, and isopropylamine; alkyl secondary amines, such as dimethylamine, diethylamine, dipropylamine, and diisopropylamine; and alkyl tertiary amines, such as trimethylamine, triethylamine, triisopropylamine, and di-N,N'-isopropylethylamine. In one embodiment, the weak organic base is an alkylamine. In one embodiment, the weak organic base is an imidazole. In one embodiment, the weak organic base is a triazole. In one embodiment, the weak organic base is pyridine. In one embodiment, the weak organic base is histidine. In one embodiment, the weak organic base is guanidine.

[0111] In one embodiment, after conjugation reaction b), the unconjugated reactive sites of the activated polysaccharide are hydrolyzed. In one embodiment, the unconjugated reactive sites are hydrolyzed by adding an aqueous solution to the conjugation solution. In one embodiment, the unconjugated reactive sites are hydrolyzed by adding an aqueous buffer solution to the conjugation solution. In one embodiment, the unconjugated reactive sites are hydrolyzed by adding an aqueous buffer solution to the conjugation solution and adjusting the pH to approximately 3.0 to approximately 10.0. In one embodiment, the unconjugated reactive sites are hydrolyzed by adding an aqueous buffer solution to the conjugation solution and adjusting the pH to approximately 7.0 to approximately 10.0. In one embodiment, the unconjugated reactive sites are hydrolyzed by adding an aqueous buffer solution to the conjugation solution and adjusting the pH to approximately 3.0 to approximately 7.0. In one embodiment, the unconjugated reactive sites are hydrolyzed by adding an aqueous buffer solution to the conjugation solution and adjusting the pH to approximately 4.0. In one embodiment, the unconjugated reactive sites are hydrolyzed by adding an aqueous buffer solution to the conjugation solution and adjusting the pH to approximately 9.0.

[0112] eTEC Chemicals In one embodiment, some or all of the pneumococcal glycoconjugates of the present invention are prepared by eTEC Chemistry, as disclosed in WO2014027302.

[0113] The eTEC spacer comprises seven straight-chain atoms (i.e., -C(O)NH(CH2)2SCH2C(O)-) and provides thioether and amide bonds that stabilize the sugar and the carrier protein. The synthesis of the eTEC-linked sugar conjugate involves a reaction between the activated hydroxyl group of the sugar and the amino group of a thioalkylamine reagent (e.g., cystamine or cysteine ​​amine or its salts) to form a carbamate bond with the sugar, providing a thiolated sugar. The generation of one or more free thiol groups is accomplished through a reaction with a reducing agent to provide an activated thiolated sugar. The free thiol group of the activated thiolated sugar reacts with an activated carrier protein containing one or more α-haloacetamide groups on an amine residue to generate a thioether bond, forming a conjugate in which the carrier protein is linked to the eTEC spacer via an amide bond.

[0114] Therefore, in one embodiment, the pneumococcal glycoconjugate of the present invention comprises a pneumococcal glycoconjugate covalently conjugated to a carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

[0115] In one embodiment, some or all of the pneumococcal glycoconjugates of the present invention comprise pneumococcal sugars bound to a carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, wherein the sugar is covalently linked to the eTEC spacer via a carbamate bond, and wherein the carrier protein is covalently linked to the eTEC spacer via an amide bond.

[0116] The eTEC-linked glycoconjugate of the present invention can be represented by general formula (III): , where (sugar) represents pneumococcal sugar.

[0117] Formula (III) is a schematic representation of the glycoconjugate of the present invention. It should not be construed as implying that only one link exists between the sugar and the carrier protein. In fact, a single carrier protein (CP) molecule can link to more than one Streptococcus pneumoniae sugar molecule, and a single sugar molecule can link to more than one single carrier protein (CP) molecule. Furthermore, most sugar repeat units remain unmodified, and a few sugar repeat units exhibit covalent linkages between the carrier protein and the sugar.

[0118] Click Chemistry In one embodiment, some or all of the pneumococcal glycoconjugates of the present invention are prepared by click chemistry (see, for example, PCT / IB2023 / 050202).

[0119] Therefore, in one embodiment, some or all of the pneumococcal glycoconjugates of the present invention comprise pneumococcal glycosides covalently conjugated to a carrier protein (CP) via a spacer and have the general formula (IV): , Where X is selected from: CH2(CH2) n’ (CH2CH2O) m CH2CH2, NHCO(CH2) n’ NHCO(CH2CH2O) m CH2CH2, OCH2(CH2) n’ and O(CH2CH2O) m CH2CH2; where n' is selected from 1 to 10 and m is selected from 1 to 4. Where X' is selected from: CH2O(CH2) n’’ CH2C=O, CH2O(CH2CH2O) m’ (CH2) n’’ CH2C=O, where n'' is selected from 0 to 10 and m' is selected from 0 to 4. The structure within square brackets represents a repeating unit of pneumococcal sugar, where n represents the number of repeating units.

[0120] In a particular aspect, the present invention relates to pneumococcal glycoconjugates comprising pneumococcal sugars covalently conjugated to a carrier protein (CP) via spacers and having the general formula (IV), wherein X is CH2 (CH2). n’ , where n' is 2, and X' is CH2O(CH2). n’’ CH2C=O, where n'' is 1.

[0121] In a particular aspect, the present invention relates to pneumococcal glycoconjugates comprising pneumococcal sugars covalently conjugated to a carrier protein (CP) via spacers and having the general formula (V). The structure within square brackets represents a repeating unit of pneumococcal sugar, where n represents the number of repeating units.

[0122] In one aspect, some or all of the pneumococcal glycoconjugates of the present invention comprise pneumococcal glycosides covalently conjugated to a carrier protein (CP) via a spacer and have the general formula (VI): , Where X is selected from: CH2(CH2) n’ (CH2CH2O) m CH2CH2, NHCO(CH2) n’ NHCO(CH2CH2O) m CH2CH2, OCH2(CH2) n’ and O(CH2CH2O) m CH2CH2; where n' is selected from 0 to 10 and m is selected from 1 to 4. Where X' is selected from: CH2(CH2) n” CH2O(CH2) n’’ CH2, CH2O (CH2CH2O) m’ (CH2) n’’ CH2, where n'' is selected from 0 to 10 and m' is selected from 0 to 4, where the structure in square brackets represents a repeating unit of pneumococcal sugar, and where n represents the number of repeating units.

[0123] In a particular aspect, the present invention relates to pneumococcal glycoconjugates comprising pneumococcal sugars covalently conjugated to a carrier protein (CP) via spacers and having the general formula (VI), wherein X is CH2 (CH2). n’ , where n' is 0, and X' is CH2(CH2). n” , where n'' is 0.

[0124] In a particular aspect, the present invention relates to pneumococcal glycoconjugates comprising pneumococcal glycosides covalently conjugated to a carrier protein (CP) via a spacer and having the general formula (VII). The structure within square brackets represents a repeating unit of pneumococcal sugar, where n represents the number of repeating units.

[0125] Formulas (IV), (V), (VII), and (VII) are schematic representations of the glycoconjugates of the present invention. It should not be construed as meaning that every repeating unit of the sugar (structure in square brackets) is linked. Instead, most sugar repeating units remain unmodified, with a few sugar repeating units showing covalent linkages between a carrier protein and the sugar. Furthermore, a single carrier protein (CP) molecule can link to more than one sugar molecule, and a single sugar molecule can link to more than one single carrier protein (CP) molecule. The structures in square brackets represent repeating units of Streptococcus pneumoniae sugars.

[0126] Following conjugation with a carrier protein, the pneumococcal glycoconjugates of the present invention can be purified (enriched relative to the amount of the glycoprotein conjugate) by various techniques known to those skilled in the art. Such techniques include dialysis, concentration / percolation, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and deep filtration. Therefore, in one embodiment, the method for producing the glycoconjugates of the present invention includes a step of purifying them after the production of the glycoconjugates.

[0127] 1.4 The glycoconjugates of Streptococcus pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 15B, 14, 18C, 19A, 19F, 22F, 23F and 33F of the present invention In one aspect, the present invention relates to compositions comprising glycoconjugates of Streptococcus pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F.

[0128] The structures of polysaccharides from Streptococcus pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F are known in the art (see, for example, Geno K et al. (2015) ClinMicrobiol Rev. Vol. 28:3, pp. 871-899).

[0129] In one embodiment, the capsular sugars of Streptococcus pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and / or 33F used in this invention are synthetic carbohydrates. The preparation of synthetic Streptococcus pneumoniae serotype 1 capsular sugars can be, for example, as disclosed in WO2015004041. The preparation of synthetic Streptococcus pneumoniae serotype 4 capsular sugars can be, for example, as disclosed in WO2016091399. The preparation of synthetic Streptococcus pneumoniae serotype 5 capsular sugars can be, for example, as disclosed in WO2016198170. The preparation of synthetic Streptococcus pneumoniae serotype 8 capsular sugars can be, for example, as disclosed in WO2017220753.

[0130] However, in a preferred embodiment, the source of the bacterial polysaccharide according to the invention may be Streptococcus pneumoniae bacterial cells. Bacterial strains that can be used as a source of Streptococcus pneumoniae capsular polysaccharide may be obtained from established culture collections (such as from a Streptococcus reference laboratory (Centers for Disease Control and Prevention, Atlanta, GA USA)) or clinical samples.

[0131] Capsular polysaccharides of Streptococcus pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F can be obtained directly from the bacteria using isolation procedures known to those skilled in the art. It can also be purchased (e.g., from the American Center for Type Culture Collection (ATCC, Manassas, VA 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)).

[0132] In cases where the capsular polysaccharide is obtained directly from bacteria, the bacterial cells can be grown in a culture medium, preferably a soybean-based medium. Following fermentation of the bacterial cells producing the Streptococcus pneumoniae capsular polysaccharide, the bacterial cells can be lysed to produce cell lysates. The capsular polysaccharide can then be separated from the cell lysates using purification techniques known in the art, including centrifugation, deep filtration, precipitation, ultrafiltration, treatment with activated carbon, percolation, and / or column chromatography (see, for example, US2006 / 0228380, US2006 / 0228381, WO2008 / 118752, and WO2020170190). The purified capsular polysaccharide can then be used to prepare glycoconjugates.

[0133] The isolated capsular sugars can be characterized by various parameters, including, for example, weight-average molecular weight (Mw).

[0134] The molecular weight of capsular sugars can be measured by a combination of size exclusion chromatography (SEC) and multi-angle laser scattering detectors (MALLS).

[0135] In one preferred embodiment, the isolated capsular polysaccharide (i.e., purified prior to further processing) has a weight-average molecular weight between 5 kDa and 5,000 kDa. In one embodiment, the isolated capsular polysaccharide has a weight-average molecular weight between 10 kDa and 3,000 kDa. In another embodiment, the isolated capsular polysaccharide has a weight-average molecular weight between 50 kDa and 1,000 kDa.

[0136] Consider any integer within any of the above ranges as an embodiment of the present invention.

[0137] To produce conjugates with favorable filterability, immunogenicity, and / or yield, the size of the capsular polysaccharide is set to the target molecular weight range prior to conjugation with the carrier protein. Advantageously, the size of purified serotypes 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 key structural features. Size setting can be performed mechanically or chemically (see, for example, WO2006 / 110381, WO2015110941).

[0138] In one embodiment, 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, propionic acid). Chemical hydrolysis can also be carried out using diluted strong acids (such as dilute hydrochloric acid, dilute sulfuric acid, dilute phosphoric acid, dilute nitric acid, or dilute perchloric acid).

[0139] The size of purified polysaccharides can also be reduced by mechanical homogenization. In one embodiment, the size of the purified polysaccharides is reduced by high-pressure homogenization. High-pressure homogenization achieves a high shear rate by pumping the process fluid through a flow path with a sufficiently small size. The shear rate is increased by using a larger applied homogenization pressure, and the exposure time can be increased by recirculating the feed stream through the homogenizer.

[0140] In one embodiment, the isolated serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F capsular polysaccharides were not size-defined.

[0141] The isolated serotype 1 capsular polysaccharide can be O-deacetylated (see, for example, WO 2008 / 079653). Therefore, in one embodiment, the isolated serotype 1 capsular polysaccharide is partially O-deacetylated. In one embodiment, O-deacetylation is performed using a weak base. Partial O-deacetylation can be performed using a sodium bicarbonate / carbonate buffer.

[0142] In one embodiment, the isolated serum-type 1 capsular polysaccharide has a weight-average molecular weight (Mw) between 100 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serum-type 1 capsular polysaccharide has a weight-average molecular weight (Mw) between 150 kDa and 900 kDa prior to conjugation. In a preferred embodiment, the isolated serum-type 1 capsular polysaccharide has a weight-average molecular weight (Mw) between 150 kDa and 700 kDa prior to conjugation. The weight-average molecular weight (Mw) of the isolated sugar prior to conjugation refers to the Mw before polysaccharide activation (i.e., after the final size setting step but before the polysaccharide reacts with the activator).

[0143] In one embodiment, the isolated serotype 4 capsular polysaccharide has a weight-average molecular weight between 100 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serotype 4 capsular polysaccharide has a weight-average molecular weight between 300 kDa and 900 kDa prior to conjugation.

[0144] In one embodiment, the isolated serum-type 5 capsular polysaccharide has a weight-average molecular weight between 100 kDa and 1,000 kDa prior to conjugation. In another embodiment, the isolated serum-type 5 capsular polysaccharide has a weight-average molecular weight between 200 kDa and 600 kDa prior to conjugation.

[0145] In one embodiment, the isolated serum-type 6A capsular polysaccharide has a weight-average molecular weight between 100 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serum-type 6A capsular polysaccharide has a weight-average molecular weight between 300 kDa and 900 kDa prior to conjugation.

[0146] In one embodiment, the isolated serum-type 6B capsular polysaccharide has a weight-average molecular weight between 100 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serum-type 6B capsular polysaccharide has a weight-average molecular weight between 200 kDa and 900 kDa prior to conjugation.

[0147] In one embodiment, the isolated serum-type 7F capsular polysaccharide has a weight-average molecular weight between 100 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serum-type 7F capsular polysaccharide has a weight-average molecular weight between 200 kDa and 900 kDa prior to conjugation.

[0148] In one embodiment, the isolated serum-type 8 capsular polysaccharide has a weight-average molecular weight between 100 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serum-type 8 capsular polysaccharide has a weight-average molecular weight between 200 kDa and 400 kDa prior to conjugation.

[0149] In one embodiment, the isolated serum-type 9V capsular polysaccharide has a weight-average molecular weight between 100 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serum-type 9V capsular polysaccharide has a weight-average molecular weight between 200 kDa and 900 kDa prior to conjugation.

[0150] In one embodiment, the isolated serum-type 10A capsular polysaccharide has a weight-average molecular weight between 100 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serum-type 10A capsular polysaccharide has a weight-average molecular weight between 200 kDa and 900 kDa prior to conjugation.

[0151] In one embodiment, the isolated serum type 11A capsular polysaccharide has a weight-average molecular weight between 100 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serum type 11A capsular polysaccharide has a weight-average molecular weight between 100 kDa and 400 kDa prior to conjugation.

[0152] In one embodiment, the isolated serum-type 12F capsular polysaccharide has a weight-average molecular weight between 100 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serum-type 12F capsular polysaccharide has a weight-average molecular weight between 150 kDa and 400 kDa prior to conjugation.

[0153] In one embodiment, the isolated serotype 14 capsular polysaccharide has a weight-average molecular weight between 100 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serotype 14 capsular polysaccharide has a weight-average molecular weight between 200 kDa and 900 kDa prior to conjugation.

[0154] In one embodiment, the isolated serum-type 15B capsular polysaccharide has a weight-average molecular weight between 100 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serum-type 15B capsular polysaccharide has a weight-average molecular weight between 150 kDa and 300 kDa prior to conjugation.

[0155] In one embodiment, the isolated serum-type 18C capsular polysaccharide has a weight-average molecular weight between 20 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serum-type 18C capsular polysaccharide has a weight-average molecular weight between 20 kDa and 500 kDa prior to conjugation.

[0156] In one embodiment, the isolated serotype 19A capsular polysaccharide has a weight-average molecular weight between 100 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serotype 19A capsular polysaccharide has a weight-average molecular weight between 250 kDa and 700 kDa prior to conjugation.

[0157] In one embodiment, the isolated serum-type 19F capsular polysaccharide has a weight-average molecular weight between 100 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serum-type 19F capsular polysaccharide has a weight-average molecular weight between 250 kDa and 800 kDa prior to conjugation.

[0158] In one embodiment, the isolated serum-type 22F capsular polysaccharide has a weight-average molecular weight between 100 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serum-type 22F capsular polysaccharide has a weight-average molecular weight between 400 kDa and 700 kDa prior to conjugation.

[0159] In one embodiment, the isolated serum-type 23F capsular polysaccharide has a weight-average molecular weight between 100 kDa and 1000 kDa prior to conjugation. In another embodiment, the isolated serum-type 23F capsular polysaccharide has a weight-average molecular weight between 200 kDa and 800 kDa prior to conjugation.

[0160] In one embodiment, the isolated serum-type 33F capsular polysaccharide has a weight-average molecular weight between 300 kDa and 2000 kDa prior to conjugation. In another embodiment, the isolated serum-type 33F capsular polysaccharide has a weight-average molecular weight between 500 kDa and 2000 kDa prior to conjugation.

[0161] In one embodiment, the serum-type 1 glycoconjugate of the present invention comprises a serum-type 1 capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0162] In one embodiment, the serum-type 4-saccharide conjugate of the present invention comprises a serum-type 4-capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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 400 kDa and 900 kDa. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0163] In one embodiment, the serum-type 5-saccharide conjugate of the present invention comprises a serum-type 5-capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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 800 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) is between 200 kDa and 500 kDa. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0164] In one embodiment, the serum-type 6A glycoconjugate of the present invention comprises a serum-type 6A capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0165] In one embodiment, the serum-type 6B glycoconjugate of the present invention comprises a serum-type 6B capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0166] In one embodiment, the serum-type 7F glycoconjugate of the present invention comprises a serum-type 7F capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0167] In one embodiment, the serum-type 8-saccharide conjugate of the present invention comprises a serum-type 8-capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0168] In one embodiment, the serum-type 9V glycoconjugate of the present invention comprises a serum-type 9V capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0169] In one embodiment, the serum-type 10A glycoconjugate of the present invention comprises a serum-type 10A capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0170] In one embodiment, the serum-type 11A glycoconjugate of the present invention comprises a serum-type 11A capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one embodiment, the weight-average molecular weight (Mw) is between 75 kDa and 600 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) is between 100 kDa and 400 kDa. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0171] In one embodiment, the serum-type 12F glycoconjugate of the present invention comprises a serum-type 12F capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0172] In one embodiment, the serum-type 14 saccharide conjugate of the present invention comprises a serum-type 14 capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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 800 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) is between 200 kDa and 600 kDa. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0173] In one embodiment, the serum-type 15B glycoconjugate of the present invention comprises a serum-type 15B capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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 600 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) is between 150 kDa and 300 kDa. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0174] In one embodiment, the serum-type 18C glycoconjugate of the present invention comprises a serum-type 18C capsular polysaccharide, wherein the weight-average molecular weight (Mw) of such polysaccharide prior to conjugation is between 20 kDa and 800 kDa. In one embodiment, the weight-average molecular weight (Mw) is between 20 kDa and 400 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) is between 20 kDa and 200 kDa. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0175] In one embodiment, the serum-type 19A glycoconjugate of the present invention comprises a serum-type 19A capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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 700 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) is between 250 kDa and 500 kDa. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0176] In one embodiment, the serum-type 19F glycoconjugate of the present invention comprises a serum-type 19F capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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 250 kDa and 600 kDa. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0177] In one embodiment, the serum-type 22F glycoconjugate of the present invention comprises a serum-type 22F capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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 400 kDa and 700 kDa. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0178] In one embodiment, the serum-type 23F glycoconjugate of the present invention comprises a serum-type 23F capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0179] In one embodiment, the serum-type 33F glycoconjugate of the present invention comprises a serum-type 33F capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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. The weight-average molecular weight (Mw) prior to conjugation refers to the Mw after polysaccharide activation (i.e., after the final size setting step and after the polysaccharide reacts with the activator).

[0180] In some embodiments, the weight-average molecular weight (Mw) of the serotype 1 glycoconjugate of the present invention is between 500 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serotype 1 glycoconjugate is between 1,000 kDa and 10,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serotype 1 glycoconjugate is between 2,000 kDa and 5,000 kDa.

[0181] In some embodiments, the weight-average molecular weight (Mw) of the serotype 4 glycoconjugate of the present invention is between 500 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serotype 4 glycoconjugate is between 1,000 kDa and 10,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serotype 4 glycoconjugate is between 2,000 kDa and 5,000 kDa.

[0182] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 5-saccharide conjugate of the present invention is between 500 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 5-saccharide conjugate is between 1,000 kDa and 10,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serum-type 5-saccharide conjugate is between 2,000 kDa and 5,000 kDa.

[0183] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 6A glycoconjugate of the present invention is between 500 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 6A glycoconjugate is between 1,000 kDa and 10,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serum-type 6A glycoconjugate is between 2,000 kDa and 5,000 kDa.

[0184] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 6B glycoconjugate of the present invention is between 500 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 6B glycoconjugate is between 1,000 kDa and 10,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serum-type 6B glycoconjugate is between 2,000 kDa and 5,500 kDa.

[0185] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 7F glycoconjugate of the present invention is between 500 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 7F glycoconjugate is between 1,000 kDa and 10,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serum-type 7F glycoconjugate is between 2,000 kDa and 5,500 kDa.

[0186] In some embodiments, the weight-average molecular weight (Mw) of the serotype 8 glycoconjugate of the present invention is between 500 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serotype 8 glycoconjugate is between 1,000 kDa and 10,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serotype 8 glycoconjugate is between 2,500 kDa and 8,000 kDa.

[0187] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 9V glycoconjugate of the present invention is between 500 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 9V glycoconjugate is between 1,000 kDa and 10,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serum-type 9V glycoconjugate is between 2,000 kDa and 5,500 kDa.

[0188] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 10A glycoconjugate of the present invention is between 500 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 10A glycoconjugate is between 1,000 kDa and 10,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serum-type 10A glycoconjugate is between 2,000 kDa and 5,500 kDa.

[0189] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 11A glycoconjugate of the present invention is between 500 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 11A glycoconjugate is between 1,000 kDa and 10,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serum-type 11A glycoconjugate is between 700 kDa and 4,500 kDa.

[0190] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 12F glycoconjugate of the present invention is between 500 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 12F glycoconjugate is between 1,000 kDa and 10,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serum-type 12F glycoconjugate is between 1,500 kDa and 4,000 kDa.

[0191] In some embodiments, the weight-average molecular weight (Mw) of the serotype 14 glycoconjugate of the present invention is between 500 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serotype 14 glycoconjugate is between 1,000 kDa and 10,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serotype 14 glycoconjugate is between 2,000 kDa and 5,500 kDa.

[0192] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 15B glycoconjugate of the present invention is between 1,000 kDa and 25,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 15B glycoconjugate is between 2,000 kDa and 20,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serum-type 15B glycoconjugate is between 5,000 kDa and 15,000 kDa.

[0193] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 18C glycoconjugate of the present invention is between 150 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 18C glycoconjugate is between 250 kDa and 7,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serum-type 18C glycoconjugate is between 300 kDa and 4,000 kDa.

[0194] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 19A glycoconjugate of the present invention is between 500 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 19A glycoconjugate is between 1,000 kDa and 10,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serum-type 19A glycoconjugate is between 2,000 kDa and 7,500 kDa.

[0195] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 19F glycoconjugate of the present invention is between 500 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 19F glycoconjugate is between 750 kDa and 10,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serum-type 19F glycoconjugate is between 1,000 kDa and 7,500 kDa.

[0196] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 22F glycoconjugate of the present invention is between 500 kDa and 10,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 22F glycoconjugate is between 1,000 kDa and 7,500 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serum-type 22F glycoconjugate is between 2,500 kDa and 5,500 kDa.

[0197] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 23F glycoconjugate of the present invention is between 500 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 23F glycoconjugate is between 750 kDa and 10,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serum-type 23F glycoconjugate is between 1,000 kDa and 7,500 kDa.

[0198] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 33F glycoconjugate of the present invention is between 500 kDa and 15,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 33F glycoconjugate is between 750 kDa and 10,000 kDa. In a preferred embodiment, the weight-average molecular weight (Mw) of the serum-type 33F glycoconjugate is between 2,000 kDa and 6,000 kDa.

[0199] The glycoconjugates of the present invention can also be characterized by the ratio (weight / weight) of sugar to carrier protein.

[0200] In some embodiments, the w / w ratio (serotype 1 polysaccharide to carrier protein) in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar 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 .

[0201] In some embodiments, the w / w ratio (of serotype 4 polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the w / w ratio is between 0.9 and 2.1. Even more preferably, the w / w ratio is between 1.0 and 1.9. In some such embodiments, the carrier protein is TT. Preferably, the carrier protein is CRM. 197 .

[0202] In some embodiments, the w / w ratio of serotype 5 polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar-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 .

[0203] In some embodiments, the w / w ratio of serum-type 6A polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar-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 .

[0204] In some embodiments, the w / w ratio of serum-type 6B polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar-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 .

[0205] In some embodiments, the w / w ratio of serum-type 7F polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar-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 .

[0206] In some embodiments, the w / w ratio of serotype 8 polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar-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 .

[0207] In some embodiments, the w / w ratio of serotype 9V polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar-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 .

[0208] In some embodiments, the w / w ratio of serum-type 10A polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar-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 .

[0209] In some embodiments, the w / w ratio (serotype 11A polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar 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 .

[0210] In some embodiments, the w / w ratio (of serum-type 12F polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar-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 .

[0211] In some embodiments, the w / w ratio (serotype 14 polysaccharide to carrier protein) in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar 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 .

[0212] In some embodiments, the w / w ratio (serotype 15B polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar 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 .

[0213] In some embodiments, the w / w ratio (of the serotype 18C polysaccharide to the carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar-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 .

[0214] In some embodiments, the w / w ratio (serotype 19A polysaccharide to carrier protein) in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar 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 .

[0215] In some embodiments, the w / w ratio (serotype 19F polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar 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 .

[0216] In some embodiments, the w / w ratio (of serotype 22F polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar-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 .

[0217] In some embodiments, the w / w ratio (of serotype 23F polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar-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 .

[0218] In some embodiments, the w / w ratio of serotype 33F polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar-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 .

[0219] Another way to characterize the glycoconjugates of the present invention is by carrier proteins (e.g., CRM). 197The number of lysine residues conjugated to the sugar in the carrier protein (DT or TT) can be characterized as the range of conjugated lysines (degree of conjugation). Evidence of lysine modification of the carrier protein (attributed to covalent linkage with the polysaccharide) can be obtained by amino acid analysis using conventional methods known to those skilled in the art. Conjugation results in a reduction in the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugated material.

[0220] In a preferred embodiment, the degree of conjugation of the serum type 1 glycoconjugate of the present invention is between 2 and 15.

[0221] In a preferred embodiment, the degree of conjugation of the serum-type 4-saccharide conjugate of the present invention is between 2 and 15.

[0222] In a preferred embodiment, the degree of conjugation of the serum-type 5-saccharide conjugate of the present invention is between 2 and 15.

[0223] In a preferred embodiment, the degree of conjugation of the serum-type 6A glycoconjugate of the present invention is between 2 and 15.

[0224] In a preferred embodiment, the degree of conjugation of the serum-type 6B glycoconjugate of the present invention is between 2 and 15.

[0225] In a preferred embodiment, the degree of conjugation of the serum-type 7F glycoconjugate of the present invention is between 2 and 15.

[0226] In a preferred embodiment, the degree of conjugation of the serum-type 8-glycoconjugate of the present invention is between 2 and 15.

[0227] In a preferred embodiment, the degree of conjugation of the serum-type 9V glycoconjugate of the present invention is between 2 and 15.

[0228] In a preferred embodiment, the degree of conjugation of the serum-type 10A glycoconjugate of the present invention is between 2 and 15.

[0229] In a preferred embodiment, the degree of conjugation of the serum-type 11A glycoconjugate of the present invention is between 2 and 15.

[0230] In a preferred embodiment, the degree of conjugation of the serum-type 12F glycoconjugate of the present invention is between 2 and 15.

[0231] In a preferred embodiment, the degree of conjugation of the serum-type 14-glycoconjugate of the present invention is between 2 and 15.

[0232] In a preferred embodiment, the degree of conjugation of the serum-type 15B glycoconjugate of the present invention is between 2 and 15.

[0233] In a preferred embodiment, the degree of conjugation of the serum-type 18C glycoconjugate of the present invention is between 2 and 15.

[0234] In a preferred embodiment, the degree of conjugation of the serum-type 19A glycoconjugate of the present invention is between 2 and 15.

[0235] In a preferred embodiment, the degree of conjugation of the serum-type 19F glycoconjugate of the present invention is between 2 and 15.

[0236] In a preferred embodiment, the degree of conjugation of the serum-type 22F glycoconjugate of the present invention is between 2 and 15.

[0237] In a preferred embodiment, the degree of conjugation of the serum-type 23F glycoconjugate of the present invention is between 2 and 15.

[0238] In a preferred embodiment, the degree of conjugation of the serum-type 33F glycoconjugate of the present invention is between 2 and 15.

[0239] The serotypes 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, and the immunogenic compositions comprising the glycoconjugates, may contain free sugars that are not covalently conjugated with the carrier protein but are still present in the glycoconjugate composition. The free sugars may non-covalently associate with the glycoconjugates (i.e., non-covalently bind to the glycoconjugates, adsorb onto the glycoconjugates, or be coated within or by the glycoconjugates).

[0240] In one embodiment, the serotype 1 glycoconjugate of the present invention comprises less than about 40% free serotype 1 polysaccharide compared to the total amount of serotype 1 polysaccharide. In a preferred embodiment, the serotype 1 glycoconjugate comprises less than about 20% free serotype 1 polysaccharide compared to the total amount of serotype 1 polysaccharide.

[0241] In one embodiment, the serotype 1 glycoconjugate of the present invention comprises less than about 40% free serotype 1 polysaccharide compared to the total amount of serotype 1 polysaccharide. In a preferred embodiment, the serotype 1 glycoconjugate comprises less than about 20% free serotype 1 polysaccharide compared to the total amount of serotype 1 polysaccharide.

[0242] In one embodiment, the serotype 4 saccharide conjugate of the present invention comprises less than about 40% free serotype 4 polysaccharide compared to the total amount of serotype 4 polysaccharide. In a preferred embodiment, the serotype 4 saccharide conjugate comprises less than about 30% free serotype 4 polysaccharide compared to the total amount of serotype 4 polysaccharide.

[0243] In one embodiment, the serotype 5 saccharide conjugate of the present invention comprises less than about 45% free serotype 5 polysaccharide compared to the total amount of serotype 5 polysaccharide. In a preferred embodiment, the serotype 5 saccharide conjugate comprises less than about 40% free serotype 5 polysaccharide compared to the total amount of serotype 5 polysaccharide.

[0244] In one embodiment, the serum-type 6A glycoconjugate of the present invention comprises less than about 40% free serum-type 6A polysaccharide compared to the total amount of serum-type 6A polysaccharide. In a preferred embodiment, the serum-type 6A glycoconjugate comprises less than about 30% free serum-type 6A polysaccharide compared to the total amount of serum-type 6A polysaccharide.

[0245] In one embodiment, the serum-type 6B glycoconjugate of the present invention comprises less than about 30% free serum-type 6B polysaccharide compared to the total amount of serum-type 6B polysaccharide. In a preferred embodiment, the serum-type 6B glycoconjugate comprises less than about 20% free serum-type 6B polysaccharide compared to the total amount of serum-type 6B polysaccharide.

[0246] In one embodiment, the serum-type 7F glycoconjugate of the present invention comprises less than about 30% free serum-type 7F polysaccharides compared to the total amount of serum-type 7F polysaccharides. In a preferred embodiment, the serum-type 7F glycoconjugate comprises less than about 20% free serum-type 7F polysaccharides compared to the total amount of serum-type 7F polysaccharides.

[0247] In one embodiment, the serotype 8 saccharide conjugate of the present invention comprises less than about 30% free serotype 8 polysaccharide compared to the total amount of serotype 8 polysaccharide. In a preferred embodiment, the serotype 8 saccharide conjugate comprises less than about 20% free serotype 8 polysaccharide compared to the total amount of serotype 8 polysaccharide.

[0248] In one embodiment, the serum-type 9V glycoconjugate of the present invention comprises less than about 40% free serum-type 9V polysaccharide compared to the total amount of serum-type 9V polysaccharide. In a preferred embodiment, the serum-type 9V glycoconjugate comprises less than about 35% free serum-type 9V polysaccharide compared to the total amount of serum-type 9V polysaccharide.

[0249] In one embodiment, the serotype 10A glycoconjugate of the present invention comprises less than about 40% free serotype 10A polysaccharide compared to the total amount of serotype 10A polysaccharide. In a preferred embodiment, the serotype 10A glycoconjugate comprises less than about 20% free serotype 10A polysaccharide compared to the total amount of serotype 10A polysaccharide.

[0250] In one embodiment, the serotype 11A glycoconjugate of the present invention comprises less than about 40% free serotype 11A polysaccharide compared 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 compared to the total amount of serotype 11A polysaccharide.

[0251] In one embodiment, the serotype 12F glycoconjugate of the present invention comprises less than about 40% free serotype 12F polysaccharide compared 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 compared to the total amount of serotype 12F polysaccharide.

[0252] In one embodiment, the serotype 14 saccharide conjugate of the present invention comprises less than about 40% free serotype 14 polysaccharide compared to the total amount of serotype 14 polysaccharide. In a preferred embodiment, the serotype 14 saccharide conjugate comprises less than about 35% free serotype 14 polysaccharide compared to the total amount of serotype 14 polysaccharide.

[0253] In one embodiment, the serotype 15B glycoconjugate of the present invention comprises less than about 40% free serotype 15B polysaccharide compared to the total amount of serotype 15B polysaccharide. In a preferred embodiment, the serotype 15B glycoconjugate comprises less than about 35% free serotype 15B polysaccharide compared to the total amount of serotype 15B polysaccharide.

[0254] In one embodiment, the serotype 18C glycoconjugate of the present invention comprises less than about 30% free serotype 18C polysaccharide compared 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 compared to the total amount of serotype 18C polysaccharide.

[0255] In one embodiment, the serotype 19A glycoconjugate of the present invention comprises less than about 40% free serotype 19A polysaccharide compared to the total amount of serotype 19A polysaccharide. In a preferred embodiment, the serotype 19A glycoconjugate comprises less than about 30% free serotype 19A polysaccharide compared to the total amount of serotype 19A polysaccharide.

[0256] In one embodiment, the serotype 19F glycoconjugate of the present invention comprises less than about 30% free serotype 19F polysaccharide compared 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 compared to the total amount of serotype 19F polysaccharide.

[0257] In one embodiment, the serotype 22F glycoconjugate of the present invention comprises less than about 40% free serotype 22F polysaccharide compared to the total amount of serotype 22F polysaccharide. In a preferred embodiment, the serotype 22F glycoconjugate comprises less than about 20% free serotype 22F polysaccharide compared to the total amount of serotype 22F polysaccharide.

[0258] In one embodiment, the serotype 23F glycoconjugate of the present invention comprises less than about 30% free serotype 23F polysaccharide compared to the total amount of serotype 23F polysaccharide. In a preferred embodiment, the serotype 23F glycoconjugate comprises less than about 20% free serotype 23F polysaccharide compared to the total amount of serotype 23F polysaccharide.

[0259] In one embodiment, the serum-type 33F glycoconjugate of the present invention comprises less than about 30% free serum-type 33F polysaccharide compared to the total amount of serum-type 33F polysaccharide. In a preferred embodiment, the serum-type 33F glycoconjugate comprises less than about 20% free serum-type 33F polysaccharide compared to the total amount of serum-type 33F polysaccharide.

[0260] Serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, or 33F glycoconjugates can also be classified by their molecular size distribution (K). d Characterization. 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-feed column to obtain a profile of the molecular size distribution of the conjugates. Large molecules excluded from the pores of the medium elute faster than small molecules. A fractionating collector is used to collect the column eluent. The fractions are colorimetrically analyzed by sugar analysis. To determine K... d The column was calibrated to establish the fraction of molecules with complete size exclusion (V0), (K d =0); and indicates the maximum retention (V) i ), (K d =1). The score that achieves the specified sample attribute (V) e ) through expression K d = (V e - V0) / (V i - V0) and K d Related.

[0261] In one embodiment, in the CL-4B column, at least 40% of the serotype 1 glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 50% of the glycoconjugates have a Kc value less than or equal to 0.3. dIn a preferred embodiment, 50% to 80% of the serotype 1 glycoconjugate is in K+ on a CL-4B column. d Less than or equal to 0.3.

[0262] In one embodiment, in the CL-4B column, at least 30% of the serotype 4 glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 40% of the glycoconjugates have a K0.3 value less than or equal to 0.3. d In a preferred embodiment, within the CL-4B column, 40% to 80% of the serotype 4-glycoconjugates have a Kc value less than or equal to 0.3. d .

[0263] In one embodiment, in the CL-4B column, at least 40% of the serotype 5-glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 55% of the glycoconjugates have a K0.3 value less than or equal to 0.3. d In a preferred embodiment, within the CL-4B column, 55% to 80% of the serum-type 5-glycoconjugates have a Kc of less than or equal to 0.3. d .

[0264] In one embodiment, in the CL-4B column, at least 50% of the serum-type 6A glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 60% of the glycoconjugates have a K0.3 or less. d In a preferred embodiment, in the CL-4B column, 60% to 90% of the serum-type 6A glycoconjugates have a Kc value less than or equal to 0.3. d .

[0265] In one embodiment, in the CL-4B column, at least 30% of the serum-type 6B glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 35% of the glycoconjugates have a K0.3 value less than or equal to 0.3. d In a preferred embodiment, within the CL-4B column, 35% to 80% of the serum-type 6B glycoconjugates have a Kc value less than or equal to 0.3. d .

[0266] In one embodiment, in the CL-4B column, at least 50% of the serum-type 7F glycoconjugates have a Kc of less than or equal to 0.3. dIn a preferred embodiment, in the CL-4B column, at least 65% of the glycoconjugates have a K0.3 value less than or equal to 0.3. d In a preferred embodiment, within the CL-4B column, 65% to 90% of the serum-type 7F glycoconjugates have a Kc value less than or equal to 0.3. d .

[0267] In one embodiment, in the CL-4B column, at least 60% of the serotype 8 glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 70% of the glycoconjugates have a K0.3 or less. d In a preferred embodiment, within the CL-4B column, 70% to 90% of the serum-type 8-glycoconjugates have a Kc value less than or equal to 0.3. d .

[0268] In one embodiment, in the CL-4B column, at least 30% of the serum-type 9V glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 40% of the glycoconjugates have a K0.3 value less than or equal to 0.3. d In a preferred embodiment, within the CL-4B column, 40% to 80% of the serum-type 9V glycoconjugates have a Kc value less than or equal to 0.3. d .

[0269] In one embodiment, in the CL-4B column, at least 40% of the serum-type 10A glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 55% of the glycoconjugates have a K0.3 value less than or equal to 0.3. d In a preferred embodiment, within the CL-4B column, 55% to 85% of the serum-type 10A glycoconjugates have a Kc value less than or equal to 0.3. d .

[0270] In one embodiment, in the CL-4B column, at least 50% of the serotype 11A glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 60% of the glycoconjugates have a K0.3 or less. d In a preferred embodiment, within the CL-4B column, 60% to 85% of the serum-type 11A glycoconjugates have a Kc value less than or equal to 0.3. d .

[0271] In one embodiment, in the CL-4B column, at least 40% of the serum-type 12F glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 50% of the glycoconjugates have a Kc value less than or equal to 0.3. d In a preferred embodiment, within the CL-4B column, 50% to 80% of the serum-type 12F glycoconjugates have a Kc value less than or equal to 0.3. d .

[0272] In one embodiment, in the CL-4B column, at least 40% of the serotype 14 glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 50% of the glycoconjugates have a Kc value less than or equal to 0.3. d In a preferred embodiment, within the CL-4B column, 50% to 80% of the serotype 14 glycoconjugates have a Kc value less than or equal to 0.3. d .

[0273] In one embodiment, in the CL-4B column, at least 30% of the serum-type 15B glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 40% of the glycoconjugates have a K0.3 value less than or equal to 0.3. d In a preferred embodiment, within the CL-4B column, 40% to 80% of the serum-type 15B glycoconjugates have a Kc value less than or equal to 0.3. d .

[0274] In one embodiment, in the CL-4B column, at least 30% of the serum-type 18C glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 40% of the glycoconjugates have a K0.3 value less than or equal to 0.3. d In a preferred embodiment, within the CL-4B column, 40% to 80% of the serum-type 18C glycoconjugates have a Kc value less than or equal to 0.3. d .

[0275] In one embodiment, in the CL-4B column, at least 40% of the serotype 19A glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 50% of the glycoconjugates have a Kc value less than or equal to 0.3. d In a preferred embodiment, within the CL-4B column, 50% to 80% of the serum-type 19A glycoconjugates have a Kc value less than or equal to 0.3.d .

[0276] In one embodiment, in the CL-4B column, at least 40% of the serotype 19F glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 50% of the glycoconjugates have a Kc value less than or equal to 0.3. d In a preferred embodiment, within the CL-4B column, 50% to 80% of the serum-type 19F glycoconjugates have a Kc value less than or equal to 0.3. d .

[0277] In one embodiment, in the CL-4B column, at least 30% of the serum-type 22F glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 40% of the glycoconjugates have a K0.3 value less than or equal to 0.3. d In a preferred embodiment, within the CL-4B column, 40% to 80% of the serum-type 22F glycoconjugates have a Kc value less than or equal to 0.3. d .

[0278] In one embodiment, in the CL-4B column, at least 30% of the serum-type 23F glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 35% of the glycoconjugates have a K0.3 value less than or equal to 0.3. d In a preferred embodiment, within the CL-4B column, 35% to 80% of the serum-type 23F glycoconjugates have a Kc value less than or equal to 0.3. d .

[0279] In one embodiment, in the CL-4B column, at least 50% of the serum-type 33F glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 60% of the glycoconjugates have a K0.3 or less. d In a preferred embodiment, within the CL-4B column, 60% to 95% of the serum-type 33F glycoconjugates have a Kc value less than or equal to 0.3. d .

[0280] In one embodiment, serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and / or 33F sugars are activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form cyanate esters. Subsequently, the activated polysaccharides are coupled directly to an amino group on a carrier protein or via a spacer group (linker). For example, the spacer group can be cystamine or cysteamine, which produces a thiolated polysaccharide that can be coupled to the carrier via a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleiminobutyryloxy]succinimide ester (GMBS)) or a halogenated acetylated carrier protein (e.g., using iodoacetamide, N-succinimide bromoacetate (SBA; SIB), N-succinimide (4-iodoacetyl)aminobenzoate (SIAB), sulfosuccinimide (4-iodoacetyl)aminobenzoate (sulfo-SIAB), N-succinimide iodoacetate (SIA), or succinimide 3-[bromoacetylamino]propionate (SBAP)). Preferably, the cyanate is coupled with hexanediamine or adipic hydrazide (ADH), and the amino-derived sugar is coupled to the carrier protein (e.g., CRM) via the carbodiimide (e.g., EDAC or EDC) chemical method through the carboxyl group on the protein carrier. 197 Conjugates. Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348 and WO 96 / 129094.

[0281] Other suitable conjugation techniques use carbodiimide, hydrazine, active ester, norbornene, 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 forming a carbonyl linker by reacting 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 reacting with a protein to form a carbamate bond. This may involve reducing the anolyte 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.

[0282] In one embodiment, the serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F glycoconjugates of the present invention are prepared using a reductive amination chemistry method. According to the present invention, reductive amination comprises two steps: (1) oxidizing (activating) the purified sugar, and (2) reducing the activated sugar and carrier protein to form a glycoconjugate (see, for example, WO2006 / 110381, WO2008 / 079653, WO2008 / 143709, WO2008 / 079732, WO2011 / 110531, WO2012 / 119972, WO2015110941, WO2015110940, WO2018 / 144439, WO2018 / 156491).

[0283] In one embodiment, the serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F glycoconjugates of the present invention are prepared using a reductive amination chemical method.

[0284] In another embodiment, the serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F and 23F glycoconjugates of the present invention are prepared using a reductive amination chemical method.

[0285] As mentioned above, the size of the isolated serotypes 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 set to a target molecular weight (MW) range before oxidation. Therefore, in one embodiment, the size of the isolated serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and / or 33F capsular polysaccharides is set prior to oxidation.

[0286] Therefore, in one embodiment, the isolated serotypes 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.

[0287] In one embodiment, isolated serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F capsular polysaccharides are conjugated to a carrier protein via a method comprising the following steps: (a) reacting the isolated serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F capsular polysaccharides with an oxidizing agent to produce activated polysaccharides; (b) mixing the activated polysaccharides from step (a) with a carrier protein; and (c) reacting the mixed activated polysaccharides and carrier protein with a reducing agent to form a glycoconjugate.

[0288] After oxidation step (a), the sugar is called activated and referred to as "activated polysaccharide".

[0289] In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes the terminal hydroxyl group to an aldehyde. In one embodiment, the oxidizing agent is periodate. For the purposes of this invention, the term "periodate" includes periodate and periodic acid; the term also includes metaperiodate (IO). 4- ) and periodate (IO6) 5- And salts including various periodic acids (such as sodium periodate and potassium periodate).

[0290] In one embodiment, isolated serotypes 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.

[0291] In one embodiment, isolated serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, and 23F capsular polysaccharides are reacted with periodate.

[0292] In a preferred embodiment, the oxidant is sodium periodate. In another embodiment, the periodate used for oxidation is metaperiodate. In a most preferred embodiment, the periodate used for oxidation is sodium metaperiodate.

[0293] When polysaccharides react with periodate, periodate oxidizes the ortho-hydroxyl group to form a carbonyl or aldehyde group, and causes C / C bond cleavage. For this reason, the term "reacting polysaccharides with periodate" includes the oxidation of the ortho-hydroxyl group by periodate.

[0294] In one embodiment, 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 polysaccharides with periodate.

[0295] In one embodiment, step a) comprises reacting serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F polysaccharides with 0.05-2 molar equivalents of periodate.

[0296] In one embodiment, step a) comprises reacting serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F polysaccharides with 0.05-0.2 molar equivalents of periodate.

[0297] In one embodiment, step a) comprises reacting serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F polysaccharides with 0.2-0.5 molar equivalents of periodate.

[0298] In one embodiment, step a) comprises reacting serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F polysaccharides with 0.5-1.5 molar equivalents of periodate.

[0299] In one embodiment, step a) comprises reacting serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F polysaccharides with 1.5-2.0 molar equivalents of periodate.

[0300] In some embodiments, the reaction in step (a) is quenched. Therefore, quenching step (a') can be performed after step (a) (see WO2015110940). Thus, in one embodiment, isolated serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and / or 33F capsular polysaccharides are conjugated to a carrier protein by a method comprising the following steps: (a) reacting the isolated serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F capsular polysaccharides with an oxidizing agent to produce activated polysaccharides; (a') quenching the oxidation reaction by adding a quenching agent to produce activated polysaccharides; (b) mixing the activated polysaccharides from step (a') with a carrier protein; and (c) reacting the mixed activated polysaccharides and carrier protein with a reducing agent to form a glycoconjugate.

[0301] In one embodiment, the oxidant is a 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical and N-chlorosuccinimide (NCS) as co-oxidants. This oxidant is particularly suitable for oxidizing serotype 12F capsular polysaccharides. In such embodiments, glycoconjugates from Streptococcus pneumoniae serotype 12F are prepared using a 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical to oxidize a primary alcohol of the sugar to an aldehyde using N-chlorosuccinimide (NCS) as a co-oxidant (hereinafter “TEMPO / NCS oxidation”), as described in Example 7 and WO 2014 / 097099. Therefore, in one aspect, the glycoconjugate from Streptococcus pneumoniae serotype 12F of the present invention can be obtained by a method 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) mixing the activated polysaccharide from step (a) with a carrier protein; and (c) reacting the mixed activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate (hereinafter “TEMPO / NCS-reductive amination”). In one aspect, the glycoconjugate from Streptococcus pneumoniae serotype 12F is obtained by this method.

[0302] In one embodiment, isolated serotypes 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.

[0303] In one embodiment, isolated serotypes 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.

[0304] In a preferred embodiment, the degree of oxidation (also referred to as the “degree of activation” in this invention document) of the activated serotypes 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.

[0305] In one embodiment, when the carrier protein is TT, the degree of oxidation of the activated serum type 1 polysaccharide is between 1 and 15 (see, for example, Table 2 of WO2019 / 152921).

[0306] In a preferred embodiment, when the carrier protein is CRM 197 At that time, the oxidation degree of activated serum type 1 polysaccharide was between 4 and 10. See Table 1 of WO2019 / 152921.

[0307] In one embodiment, the oxidation degree of the activated serum type 4 polysaccharide is between 1 and 15. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation level of activated serum type 4 polysaccharide was between 1 and 5. See Table 1 of WO2019 / 152921.

[0308] In one embodiment, the oxidation degree of the activated serum type 5 polysaccharide is between 1 and 15. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation level of activated serum type 5 polysaccharide was between 2 and 6. See Table 1 of WO2019 / 152921.

[0309] In one embodiment, when the carrier protein is TT, the oxidation level of the activated serum type 5 polysaccharide is between 1 and 15. See Table 2 of WO2019 / 152921.

[0310] In one embodiment, the oxidation degree of the activated serum-type 6A polysaccharide is between 1 and 15. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation level of activated serum type 6A polysaccharide was between 5 and 15. See Table 1 of WO2019 / 152921.

[0311] In one embodiment, the oxidation degree of the activated serum-type 6B polysaccharide is between 1 and 15. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation level of activated serum type 6B polysaccharide was between 7 and 13. See Table 1 of WO2019 / 152921.

[0312] In one embodiment, the oxidation degree of the activated serum-type 7F polysaccharide is between 15. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation degree of activated serum-type 7F polysaccharide was between 2 and 8. See Table 1 of WO2019 / 152921.

[0313] In one embodiment, the oxidation degree of the activated serum type 8 polysaccharide is between 1 and 20. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation degree of activated serum type 8 polysaccharide was between 1 and 17. See Table 1 of WO2019 / 152921.

[0314] In one embodiment, the oxidation degree of the activated serum-type 9V polysaccharide is between 1 and 15. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation level of activated serum-type 9V polysaccharide was between 4 and 9. See Table 1 of WO2019 / 152921.

[0315] In one embodiment, the oxidation degree of the activated serum-type 10A polysaccharide is between 1 and 15. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation degree of activated serum-type 10A polysaccharide was between 1 and 12. See Table 1 of WO2019 / 152921.

[0316] In one embodiment, the oxidation degree of the activated serum-type 11A polysaccharide is between 1 and 20. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation degree of activated serum type 11A polysaccharide was between 1 and 15. See Table 1 of WO2019 / 152921.

[0317] In one embodiment, the oxidation degree of the activated serum-type 12F polysaccharide is between 1 and 15. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation level of activated serum-type 12F polysaccharide was between 1 and 9. See Table 1 of WO2019 / 152921.

[0318] In one embodiment, the oxidation degree of the activated serum-type 14 polysaccharide is between 1 and 20. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation degree of activated serum type 14 polysaccharide was between 6 and 13. See Table 1 of WO2019 / 152921.

[0319] In one embodiment, the oxidation degree of the activated serum-type 15B polysaccharide is between 1 and 20. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation degree of activated serum-type 15B polysaccharide was between 1 and 17. See Table 1 of WO2019 / 152921.

[0320] In one embodiment, when the carrier protein is TT, the oxidation level of the activated serum-type 15B polysaccharide is between 1 and 15. See Table 2 of WO2019 / 152925.

[0321] In one embodiment, the oxidation degree of the activated serum-type 18C polysaccharide is between 1 and 20. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation degree of activated serum-type 18C polysaccharide was between 6 and 14. See Table 1 of WO2019 / 152921.

[0322] In one embodiment, the oxidation level of the activated serum-type 19A polysaccharide is between 1 and 20. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation level of activated serum type 19A polysaccharide was between 7 and 13. See Table 1 of WO2019 / 152921.

[0323] In one embodiment, the oxidation degree of the activated serum-type 19F polysaccharide is between 1 and 20. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation level of activated serum-type 19F polysaccharide was between 6 and 12. See Table 1 of WO2019 / 152921.

[0324] In one embodiment, the oxidation degree of the activated serum-type 22F polysaccharide is between 1 and 20. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation degree of activated serum-type 22F polysaccharide was between 1 and 16. See Table 1 of WO2019 / 152921.

[0325] In one embodiment, when the carrier protein is TT, the oxidation level of the activated serum-type 22F polysaccharide is between 1 and 20. See Table 2 of WO2019 / 152925.

[0326] In one embodiment, the oxidation degree of the activated serum-type 23F polysaccharide is between 1 and 20. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation degree of activated serum-type 23F polysaccharide was between 6 and 14. See Table 1 of WO2019 / 152921.

[0327] In one embodiment, the oxidation degree of the activated serum-type 33F polysaccharide is between 1 and 20. In a preferred embodiment, when the carrier protein is CRM... 197 At that time, the oxidation degree of activated serum-type 33F polysaccharide was between 1 and 15. See Table 1 of WO2019 / 152921.

[0328] The activated polysaccharide and carrier protein can be lyophilized, either independently (discrete lyophilization) or together (co-lyophilization). In one embodiment, the activated polysaccharide and carrier protein are co-lyophilized. In another embodiment, the activated polysaccharide and carrier protein are lyophilized independently.

[0329] In one embodiment, freeze drying occurs in the presence of non-reducing sugars, which may include sucrose, trehalose, raffinose, stachyose, melitriose, dextran, mannitol, lactitol, and isomaltitol.

[0330] In one embodiment, the initial input ratio (by weight) of activated serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and / or 33F capsular polysaccharides to carrier proteins at step b) is between 4:1 and 0.1:1. In another embodiment, the initial input ratio (by weight) of activated serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and / or 33F capsular polysaccharides to carrier proteins is between 1.5:1 and 0.5:1.

[0331] In one embodiment, reduction reaction (c) is carried out in an aprotic solvent. In one embodiment, reduction reaction (c) is carried out in a solution consisting essentially of dimethyl sulfoxide (DMSO). In one embodiment, reduction reaction (c) is carried out in DMSO (dimethyl sulfoxide) solvent.

[0332] In one embodiment, the reduction reaction (c) is carried out in an aqueous solvent.

[0333] In one embodiment, reduction reaction (c) is carried out in DMSO (dimethyl sulfoxide) solvent of serum types 6A, 6B, 7F, 8, 10A, 15B, 19A, 19F, 22F and 23F, and reduction reaction (c) is carried out in aqueous solvent of serum types 1, 4, 5, 9V, 11A, 12F, 14 and 18C.

[0334] In one embodiment, reduction reaction (c) is carried out in DMSO (dimethyl sulfoxide) solvent of serum types 6A, 6B, 7F, 8, 10A, 15B, 19A, 19F, 22F and 23F, and reduction reaction (c) is carried out in aqueous solvent of serum types 1, 4, 5, 9V, 11A, 12F, 14, 18C and 33F.

[0335] In one embodiment, reduction reaction (c) is carried out in DMSO (dimethyl sulfoxide) solvent of serum types 6A, 6B, 7F, 18C, 19A, 19F and 23F, and reduction reaction (c) is carried out in aqueous solvent of serum types 1, 4, 5, 9V, 14, 22F and 33F.

[0336] In one embodiment, reduction reaction (c) is carried out in DMSO (dimethyl sulfoxide) solvent of serum types 6A, 6B, 7F, 11A, 12F, 19A, 19F and 23F, and reduction reaction (c) is carried out in aqueous solvent of serum types 1, 4, 5, 8, 9V, 10A, 14, 15B, 18C, 22F and 33F.

[0337] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride or zinc borohydride in the presence of Bronsted acid or Lewis acid, or amine boranes such as pyridineborane, 2-methylpyridineborane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-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).

[0338] In one embodiment, a reducing agent in a amount between 0.2 and 10 molar equivalents is used in step c). Preferably, a reducing agent in a amount between 0.5 and 5 molar equivalents is used in step c).

[0339] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate, which can be capped using a suitable capping agent. In one embodiment, the capping agent is sodium borohydride (NaBH4).

[0340] In one embodiment, end-capping is achieved by mixing the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In another embodiment, end-capping is achieved by mixing the product of step c) with 1 to 3 molar equivalents of sodium borohydride.

[0341] Following conjugation with a carrier protein, serotypes 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 (enriched relative to the amount of the glycoprotein conjugate) using a variety of techniques known to those skilled in the art. Such techniques include dialysis, concentration / percolation, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and deep filtration. Therefore, in one embodiment, the method for generating the serotype 1 glycoconjugate of the present invention includes a step of purifying the glycoconjugate after its generation.

[0342] In some embodiments, the serum-type 33F glycoconjugate of the present invention is prepared using reductive amination.

[0343] In other embodiments, the serum-type 33F glycoconjugates of the present invention are prepared using eTEC conjugation (hereinafter “serum-type 33F eTEC-linked glycoconjugates”), such as that described in WO 2014 / 027302 or WO2015110941 (see Examples 1, 2, and 3). Such 33F glycoconjugates comprise a sugar covalently conjugated to a carrier protein via one or more eTEC spacer groups, wherein the sugar is covalently conjugated to the eTEC spacer group via a carbamate bond, and wherein the carrier protein is covalently conjugated to the eTEC spacer group via an amide bond. The eTEC-linked glycoconjugates of the present invention can be represented by the following general formula (III): The atoms containing the eTEC spacer group are enclosed in the central box.

[0344] The eTEC spacer group comprises seven linear atoms (i.e., -C(O)NH(CH2)2SCH2C(O)-) and provides stable thioether and amide bonds between the sugar and the carrier protein. The synthesis of the eTEC-linked sugar conjugate involves the reaction of the activated hydroxyl group of the sugar with the amino group of a thioalkylamine reagent (e.g., cystamine or cysteine ​​or a salt thereof) to form a carbamate bond with the sugar, providing a thiolated sugar. The generation of one or more free thiohydrogen groups is achieved by reacting with a reducing agent to provide an activated thiolated sugar. The reaction of the free thiohydrogen group of the activated thiolated sugar with an activated carrier protein having one or more α-haloacetamide residues on an amine-containing residue produces a thioether bond to form a conjugate, wherein the carrier protein is linked to the eTEC spacer group via an amide bond.

[0345] In the serotype 33F eTEC-linked glycoconjugates of the present invention, the sugar may be a polysaccharide or an oligosaccharide. The carrier protein may be selected from any suitable carrier as described herein or known to those skilled in the art. Preferably, the sugar is a polysaccharide. In some such embodiments, the carrier protein is CRM. 197In some such embodiments, the eTEC-linked glycoconjugate comprises Streptococcus pneumoniae serotype 33F capsular polysaccharide.

[0346] In a particularly preferred embodiment, the eTEC-linked glycoconjugate comprises Pn-33F capsular polysaccharide, which is covalently linked to CRM via an eTEC spacer group (serum-type 33F eTEC-linked glycoconjugate). 197 .

[0347] In one embodiment, the serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, and 23F glycoconjugates of the present invention are prepared using a reductive amination chemical method, and the serotype 33F glycoconjugate of the present invention is prepared using eTEC conjugation.

[0348] 1.5 The Streptococcus pneumoniae serotype 15A glycoconjugate of the present invention In one aspect, the present invention relates to Streptococcus pneumoniae serotype 15A glycoconjugates.

[0349] The structure of the serotype 15A polysaccharide of Streptococcus pneumoniae is known in the art (see, for example, Geno K et al. (2015) Clin Microbiol Rev 28:3, pp. 871-899).

[0350] In one embodiment, the Streptococcus pneumoniae serotype 15A sugar used in this invention is a synthetic carbohydrate.

[0351] However, in a preferred embodiment, the source of the bacterial polysaccharide according to the invention may be Streptococcus pneumoniae serotype 15A bacterial cells. Bacterial strains that can be used as a source of Streptococcus pneumoniae serotype 15A polysaccharide may be obtained from an established culture collection (such as from a streptococcal reference laboratory (Centers for Disease Control and Prevention, Atlanta, GA USA)) or clinical samples.

[0352] Serotype 15A sugar can be obtained directly from bacteria using isolation procedures known to those skilled in the art. It can also be purchased (e.g., from the United States Type Culture Collection (ATCC, Manassas, VA USA) (e.g., reference number ATCC(537-X)).

[0353] In cases where serotype 15A sugar is obtained directly from bacteria, the bacterial cells are preferably grown in a soybean-based medium. Following fermentation of the bacterial cells producing Streptococcus pneumoniae serotype 15A capsular polysaccharide, the bacterial cells can be lysed to produce cell lysates. The serotype 15A polysaccharide can then be separated from the cell lysates using purification techniques known in the art, including centrifugation, deep filtration, precipitation, ultrafiltration, treatment with activated carbon, percolation, and / or column chromatography (see, for example, US2006 / 0228380, US2006 / 0228381, WO2008 / 118752, and WO2020170190). The purified serotype 15a capsular polysaccharide can then be used to prepare glycoconjugates.

[0354] The isolated serum type 15A capsular sugar obtained by purifying serum type 15A polysaccharide from Streptococcus pneumoniae lysate and optionally setting the size of the purified polysaccharide can be characterized by various parameters, including, for example, weight average molecular weight (Mw).

[0355] The molecular weight of polysaccharides can be measured by combining size exclusion chromatography (SEC) with multi-angle laser scattering detectors (MALLS).

[0356] In one preferred embodiment, the isolated serum-type 15A capsular polysaccharide (i.e., purified prior to further processing) has a weight-average molecular weight between 100 kDa and 2500 kDa. In one embodiment, the isolated serum-type 15A capsular polysaccharide has a weight-average molecular weight between 250 kDa and 1500 kDa. In one embodiment, the isolated serum-type 15A capsular polysaccharide has a weight-average molecular weight between 500 kDa and 1000 kDa.

[0357] Consider any integer within any of the above ranges as an embodiment of the present invention.

[0358] To produce serum-type 15A conjugates with favorable filterability, immunogenicity, and / or yield, the size of the polysaccharide is set to a target molecular weight range prior to conjugation with the carrier protein. Advantageously, the purified serum-type 15A polysaccharide has a reduced size while retaining key structural features. Size setting can be performed mechanically or chemically.

[0359] In one embodiment, the size of the purified serum-type 15A polysaccharide is reduced by chemical hydrolysis. Chemical hydrolysis can be carried out using a weak acid (e.g., acetic acid, formic acid, propionic acid). Chemical hydrolysis can also be carried out using diluted strong acids (such as dilute hydrochloric acid, dilute sulfuric acid, dilute phosphoric acid, dilute nitric acid, or dilute perchloric acid).

[0360] However, preferably, the size of the purified serum-type 15A polysaccharide is reduced by mechanical homogenization. In one embodiment, the size of the purified serum-type 15A polysaccharide is reduced by high-pressure homogenization. High-pressure homogenization achieves a high shear rate by pumping the process fluid through a flow path with a sufficiently small size. The shear rate is increased by using a larger applied homogenization pressure, and the exposure time can be increased by recirculating the feed stream through the homogenizer.

[0361] In one embodiment, the isolated serum-type 15A capsular polysaccharide is sized to have a weight-average molecular weight between 50 kDa and 500 kDa. In a preferred embodiment, the isolated serum-type 15A capsular polysaccharide is sized to have a weight-average molecular weight between 75 kDa and 250 kDa. Preferably, the isolated serum-type 15A capsular polysaccharide is sized to have a weight-average molecular weight below 175 kDa. In an even more preferred embodiment, the isolated serum-type 15A capsular polysaccharide is sized to have a weight-average molecular weight between 75 kDa and 175 kDa. In a most preferred embodiment, the isolated serum-type 15A capsular polysaccharide is sized to have a weight-average molecular weight between 100 kDa and 175 kDa. Preferably, the isolated serum-type 15A polysaccharide is sized by mechanical homogenization, preferably by high-pressure homogenization.

[0362] In one embodiment, the isolated serum-type 15A capsular polysaccharide was not size-set.

[0363] In one embodiment, the isolated serotype 15A capsular polysaccharide has a weight-average molecular weight between 50 kDa and 500 kDa prior to conjugation. In another embodiment, the isolated serotype 15A capsular polysaccharide has a weight-average molecular weight between 75 kDa and 250 kDa prior to conjugation. In a preferred embodiment, the isolated serotype 15A capsular polysaccharide has a weight-average molecular weight between 75 kDa and 175 kDa prior to conjugation. In an even more preferred embodiment, the isolated serotype 15A capsular polysaccharide has a weight-average molecular weight between 90 kDa and 150 kDa prior to conjugation. In a most preferred embodiment, the isolated serotype 15A capsular polysaccharide has a weight-average molecular weight between 100 kDa and 175 kDa prior to conjugation.

[0364] The weight-average molecular weight (Mw) of the sugar prior to conjugation refers to the Mw before polysaccharide activation (i.e., after the final size setting step but before the polysaccharide reacts with the activator). In the case of this invention, the Mw of the 15A polysaccharide is substantially unchanged by the activation step, and the Mw of the 15A polysaccharide incorporated into the conjugate is similar to the Mw of the polysaccharide measured prior to activation.

[0365] In one embodiment, the serum-type 15A glycoconjugate of the present invention comprises a serum-type 15A capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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.

[0366] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 15A glycoconjugate of the present invention is between 500 kDa and 10,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 15A glycoconjugate is between 1,000 kDa and 10,000 kDa. Preferably, the weight-average molecular weight (Mw) of the serum-type 15A glycoconjugate is between 1,000 kDa and 6,000 kDa.

[0367] The serotype 15A glycoconjugate of the present invention can also be characterized by the ratio (w / w) of sugar to carrier protein. In some embodiments, the ratio (w / w) of serotype 15A polysaccharide to carrier protein in the glycoconjugate is between 0.5 and 3.0. Preferably, the sugar to carrier protein ratio (w / w) is between 0.5 and 1.5. Even more preferably, the sugar to carrier protein ratio (w / w) is between 0.7 and 1.1.

[0368] Another way to characterize the serum-type 15A glycoconjugate of the present invention is by carrier protein (e.g., CRM). 197 The number of lysine residues conjugated to the sugar in the carrier protein (DT or TT) can be characterized as the range of conjugated lysine residues (degree of conjugation). Evidence of lysine modification of the carrier protein (attributed to covalent linkage with the polysaccharide) can be obtained by amino acid analysis and using conventional methods known to those skilled in the art. Conjugation results in a reduction in the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugated material. In a preferred embodiment, the degree of conjugation of the serotype 15A glycoconjugate of the present invention is between 2 and 20. Preferably, the degree of conjugation of the serotype 15A glycoconjugate of the present invention is between 5 and 10.

[0369] The serum type 15A glycoconjugate and immunogenic composition of the present invention may contain free sugars that are not covalently conjugated with the carrier protein but are still present in the glycoconjugate composition. The free sugars may non-covalently associate with the glycoconjugate (i.e., non-covalently bind to the glycoconjugate, adsorb to the glycoconjugate, or be coated in or by the glycoconjugate).

[0370] In one embodiment, the serotype 15A glycoconjugate comprises less than about 40% free serotype 15A polysaccharide compared 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 compared to the total amount of serotype 15A polysaccharide.

[0371] Serum-type 15A glycoconjugates can also be classified by their molecular size distribution (K). d Characterization. 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-feed column to obtain a profile of the molecular size distribution of the conjugates. Large molecules excluded from the pores of the medium elute faster than small molecules. An elution fractionator is used to collect the column eluent. The elution fractions are colorimetrically analyzed by sugar analysis. To determine K... d The column was calibrated to establish the fraction of molecules with complete size exclusion (V0), (K d =0); and indicates the maximum retention (V) i ), (K d =1). The score that achieves the specified sample attribute (V) e ) through expression K d = (V e - V0) / (V i - V0) and K d Related.

[0372] In one embodiment, in the CL-4B column, at least 40% of the serum-type 15A glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 50% of the glycoconjugates have a Kc value less than or equal to 0.3. d In a preferred embodiment, between 50% and 90% of the serum-type 15A glycoconjugate is in K+ on a CL-4B column. d Less than or equal to 0.3.

[0373] In one embodiment, the serotype 15A sugar is activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. Subsequently, the activated polysaccharide is directly coupled with a carrier protein (preferably CRM). 197The amino group on the carrier is coupled to the carrier via a spacer group (linker). For example, the spacer group may be cystamine or cysteamine, which produces a thiolated polysaccharide. This thiolated polysaccharide may be coupled to the carrier via a thioether bond obtained after reacting with a maleimide-activated carrier protein (e.g., using N-[γ-maleiminobutyryloxy]succinimide ester (GMBS)) or a halogenated acetylated carrier protein (e.g., using iodoacetamide, N-succinimide bromoacetate (SBA; SIB), N-succinimide (4-iodoacetyl)aminobenzoate (SIAB), sulfosuccinimide (4-iodoacetyl)aminobenzoate (sulfo-SIAB), N-succinimide iodoacetate (SIA), or succinimide 3-[bromoacetylamino]propionate (SBAP)). Preferably, the cyanate is coupled with hexanediamine or adipic hydrazide (ADH), and the amino-derived sugar is coupled to the carrier protein (e.g., CRM) via the carbodiimide (e.g., EDAC or EDC) chemical method through the carboxyl group on the protein carrier. 197 Conjugates. Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348 and WO 96 / 129094.

[0374] Other suitable conjugation techniques use carbodiimide, hydrazine, active ester, norbornene, 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 forming a carbonyl linker by reacting 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 reacting with a protein to form a carbamate bond. This may involve reducing the anolyte 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.

[0375] In a preferred embodiment, the serum-type 15A glycoconjugate of the present invention is prepared using a reductive amination chemical method. According to the present invention, reductive amination comprises two steps: (1) oxidizing (activating) the purified sugar, and (2) reducing the activated sugar and carrier protein to form the glycoconjugate (see, for example, WO2006 / 110381, WO2008 / 079653, WO2008 / 143709, WO2008 / 079732, WO2011 / 110531, WO2012 / 119972, WO2015110941, WO2015110940, WO2018 / 144439, WO2018 / 156491).

[0376] In one embodiment, the serum type 15A glycoconjugate of the present invention is prepared by a method comprising the following steps: conjugating isolated serum type 15A capsular polysaccharide with a carrier protein. (a) reacting the isolated serum type 15A capsular polysaccharide with an oxidizing agent; (b) mixing the activated polysaccharide from step (a) with a carrier protein; and (c) reacting the mixed activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate.

[0377] As mentioned above, the size of the isolated serum-type 15A capsular polysaccharide can be set to a target molecular weight (MW) range prior to oxidation. Therefore, in one embodiment, the isolated serum-type 15A capsular polysaccharide is size-set prior to oxidation.

[0378] In one embodiment, the isolated serum-type 15A capsular polysaccharide was not size-set prior to oxidation.

[0379] In one embodiment, oxidation step (a) is carried out at a pH between 4.0 and 6.0. Preferably, oxidation step (a) is carried out at a pH between 4.5 and 5.5.

[0380] In one embodiment, the oxidation step (a) is carried out at a pH of about 5.0.

[0381] After oxidation step (a), the sugar is called activated and referred to as "activated polysaccharide".

[0382] In one embodiment, the activated serum-type 15A polysaccharide of the present invention has a weight-average molecular weight (Mw) between 50 kDa and 500 kDa. In another 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.

[0383] In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes the terminal hydroxyl group to an aldehyde. In one embodiment, the oxidizing agent is periodate. For the purposes of this invention, the term "periodate" includes periodate and periodic acid; the term also includes metaperiodate (IO). 4- ) and periodate (IO6) 5- And salts including various periodic acids (such as sodium periodate and potassium periodate).

[0384] In a preferred embodiment, the oxidant is sodium periodate. In another embodiment, the periodate used for oxidation is metaperiodate. In a most preferred embodiment, the periodate used for oxidation is sodium metaperiodate.

[0385] When polysaccharides react with periodate, periodate oxidizes the ortho-hydroxyl group to form a carbonyl or aldehyde group, and causes C / C bond cleavage. For this reason, the term "reacting polysaccharides with periodate" includes the oxidation of the ortho-hydroxyl group by periodate.

[0386] In one embodiment, 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.

[0387] In one embodiment, the degree of oxidation of the activated serum-type 15A polysaccharide (also referred to as the "degree of activation" in this invention document) is between 2 and 20. In a preferred embodiment, the degree of oxidation of the activated serum-type 15A polysaccharide is between 2 and 8. In a most preferred embodiment, the degree of oxidation of the activated serum-type 15A polysaccharide is 5 ± 2.5.

[0388] In one embodiment, the activated serotype 15A polysaccharide and carrier protein are lyophilized before step b). Preferably, lyophilization is performed after step a). In one embodiment, the activated polysaccharide is lyophilized after step a) and the carrier protein is also lyophilized. In one embodiment, the activated serotype 15A 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.

[0389] In one embodiment, the activated serotype 15A polysaccharide and the carrier protein are lyophilized independently (discrete lyophilization). In another embodiment, the activated serotype 15A polysaccharide and the carrier protein are lyophilized together (co-lyophilization).

[0390] In one embodiment, freeze-drying occurs in the presence of non-reducing sugars, which may include sucrose, trehalose, raffinose, stachyose, melitriose, dextran, mannitol, lactitol, and isomaltitol. In one embodiment, the sugar is selected from the group consisting of sucrose, trehalose, and mannitol. In one embodiment, the sugar is sucrose, trehalose, or mannitol. In one embodiment, the sugar is sucrose.

[0391] In one embodiment, the initial input ratio (by weight) of the activated serotype 15A capsular polysaccharide to the carrier protein at step b) is between 3:1 and 0.5:1. In another embodiment, the initial input ratio (by weight) of the activated serotype 15A capsular polysaccharide to the carrier protein is between 1.5:1 and 0.5:1. Preferably, the initial input ratio (by weight) of the activated serotype 15A capsular polysaccharide to the carrier protein is between 1.1:1 and 0.9:1.

[0392] 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.

[0393] 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).

[0394] In one embodiment, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent.

[0395] Most preferably, the reduction reaction (c) is carried out in DMSO (dimethyl sulfoxide) solvent.

[0396] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride or zinc borohydride in the presence of Bronsted acid or Lewis acid, or amine boranes such as pyridineborane, 2-methylpyridineborane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3, or 5-ethyl-2-methylpyridineborane (PEMB). 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.

[0397] In one embodiment, a reducing agent in a amount between 0.2 and 5 molar equivalents is used in step c). Preferably, a reducing agent in a amount between 0.5 and 2 molar equivalents is used in step c). Most preferably, a reducing agent in a amount between 0.9 and 1.1 molar equivalents is used in step c).

[0398] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate, which can be capped using a suitable capping agent. In one embodiment, the capping agent is sodium borohydride (NaBH4).

[0399] In one embodiment, end-capping is achieved by mixing the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In another embodiment, end-capping is achieved by mixing the product of step c) with 1 to 3 molar equivalents of sodium borohydride.

[0400] Following conjugation with a carrier protein, the serum-type 15A glycoconjugate can be purified (enriched relative to the amount of the glycoprotein conjugate) using a variety of techniques known to those skilled in the art. Such techniques include dialysis, concentration / percolation, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and deep filtration. Therefore, in one embodiment, the method for generating the serum-type 15A glycoconjugate of the present invention includes a step of purifying the glycoconjugate after its generation.

[0401] 1.6 The pneumococcal serotype 23A glycoconjugate of the present invention In one aspect, the present invention relates to Streptococcus pneumoniae serotype 23A glycoconjugates.

[0402] The structure of the serotype 23A polysaccharide is known in the art (see, for example, Ravenscroft N et al. (2017) Carbohydrate Res. Vol. 450, pp. 19-29 and WO2019050814). The structure of the serotype 23A capsular polysaccharide is: →4)-β-D-Glc p -(1→3)-[[α-L-Rha p -(1→2)]-[Gro-(2→P→3)]-β-D-Gal p -(1→4)]-β-L-Rha p -(1→).

[0403] In one embodiment, the Streptococcus pneumoniae serotype 23A sugar used in this invention is a synthetic carbohydrate.

[0404] However, in a preferred embodiment, the source of the bacterial polysaccharide according to the invention may be Streptococcus pneumoniae serotype 23A bacterial cells. Bacterial strains that can be used as a source of Streptococcus pneumoniae serotype 23A polysaccharide may be obtained from an established culture collection (such as from a streptococcal reference laboratory (Centers for Disease Control and Prevention, Atlanta, GA USA)) or clinical samples.

[0405] Serotype 23A sugar can be obtained directly from bacteria using isolation procedures known to those skilled in the art. It can also be purchased (e.g., from the United States Type Culture Collection (ATCC, Manassas, VA USA) (e.g., references ATCC 545-X, ATCC 546-X, ATCC 547-X)).

[0406] In cases where serotype 23A sugar is obtained directly from bacteria, the bacterial cells can be grown in a culture medium, preferably a soybean-based medium. Following fermentation of the bacterial cells producing the pneumococcal serotype 23A capsular polysaccharide, the bacterial cells can be lysed to produce cell lysates. The serotype 23A polysaccharide can then be separated from the cell lysates using purification techniques known in the art, including centrifugation, deep filtration, precipitation, ultrafiltration, treatment with activated carbon, percolation, and / or column chromatography (see, for example, US2006 / 0228380, US2006 / 0228381, WO2008 / 118752, and WO2020170190). The purified serotype 23A capsular polysaccharide can then be used to prepare glycoconjugates.

[0407] The isolated serum type 23A capsular sugar obtained by purifying serum type 23A polysaccharide from Streptococcus pneumoniae lysate and optionally setting the size of the purified polysaccharide can be characterized by various parameters, including, for example, weight average molecular weight (Mw).

[0408] The molecular weight of polysaccharides can be measured by combining size exclusion chromatography (SEC) with multi-angle laser scattering detectors (MALLS).

[0409] In one preferred embodiment, the isolated serum-type 23A capsular polysaccharide (i.e., purified prior to further processing) has a weight-average molecular weight between 100 kDa and 2500 kDa. In one embodiment, the isolated serum-type 23A capsular polysaccharide has a weight-average molecular weight between 250 kDa and 1500 kDa. In one embodiment, the isolated serum-type 23A capsular polysaccharide has a weight-average molecular weight between 500 kDa and 1000 kDa.

[0410] Consider any integer within any of the above ranges as an embodiment of the present invention.

[0411] To produce serum-type 23A conjugates with favorable filterability, immunogenicity, and / or yield, the size of the polysaccharide is set to a target molecular weight range prior to conjugation with the carrier protein. Advantageously, the purified serum-type 23A polysaccharide has a reduced size while retaining key structural features. Size setting can be performed mechanically or chemically.

[0412] Most preferably, the size of the purified serum-type 23A polysaccharide is reduced by mechanical homogenization.

[0413] It has been found that the use of acid hydrolysis recommended, for example, in WO2019050814 or WO2019050818, is not suitable for reducing the size of serum type 23A polysaccharide.

[0414] Acid hydrolysis has been found to affect the structural integrity of serum-type 23A polysaccharide. Even relatively mild hydrolysis (e.g., treatment with 100 mM acetic acid at 80°C for 2 hours) has been found to cause a 25% loss of branched rhamnose (α-L-Rhap-(1→2)) (see [link to original text]). Figure 1 ).

[0415] Mechanically sized residues allow for the preservation of residues, which may be important from an immunological perspective. Furthermore, this residue is the primary activation site for periodate oxidation. Therefore, retaining this residue is crucial in the context of periodate oxidation using polysaccharides (e.g., the activation step in reductive amination chemical processes).

[0416] Therefore, in a preferred embodiment, the size of the purified serum-type 23A polysaccharide is reduced by mechanical homogenization.

[0417] In one embodiment, the size of the purified serum-type 23A polysaccharide is reduced by high-pressure homogenization. High-pressure homogenization achieves a high shear rate by pumping the process fluid through a flow path with a sufficiently small size. The shear rate is increased by using a larger applied homogenization pressure, and the exposure time can be increased by recirculating the feed stream through the homogenizer.

[0418] In a preferred embodiment, the method for preparing the serum type 23A glycoconjugate of the present invention does not include the step of determining the serum type 23A polysaccharide by acid hydrolysis.

[0419] In one embodiment, the isolated serum-type 23A capsular polysaccharide is sized to have a weight-average molecular weight between 50 kDa and 500 kDa. In a preferred embodiment, the isolated serum-type 23A capsular polysaccharide is sized to have a weight-average molecular weight between 75 kDa and 400 kDa. In an even more preferred embodiment, the isolated serum-type 23A capsular polysaccharide is sized to have a weight-average molecular weight between 100 kDa and 350 kDa. In a most preferred embodiment, the isolated serum-type 23A capsular polysaccharide is sized to have a weight-average molecular weight between 125 kDa and 225 kDa. Preferably, the isolated serum-type 23A polysaccharide is sized by mechanical homogenization, preferably by high-pressure homogenization.

[0420] In one embodiment, the isolated serum type 23A capsular polysaccharide was not size-set.

[0421] In one embodiment, the isolated serotype 23A capsular polysaccharide has a weight-average molecular weight between 50 kDa and 500 kDa prior to conjugation. In another embodiment, the isolated serotype 23A capsular polysaccharide has a weight-average molecular weight between 75 kDa and 400 kDa prior to conjugation. In an even more preferred embodiment, the isolated serotype 23A capsular polysaccharide has a weight-average molecular weight between 100 kDa and 350 kDa prior to conjugation. In a most preferred embodiment, the isolated serotype 23A capsular polysaccharide has a weight-average molecular weight between 125 kDa and 225 kDa prior to conjugation.

[0422] The weight-average molecular weight (Mw) of the sugar prior to conjugation refers to the Mw before polysaccharide activation (i.e., after the final size setting step but before the polysaccharide reacts with the activator). In the case of this invention, the Mw of the 23A polysaccharide is substantially unchanged by the activation step, and the Mw of the 23A polysaccharide incorporated into the conjugate is similar to the Mw of the polysaccharide measured prior to activation.

[0423] In one embodiment, the serum-type 23A glycoconjugate of the present invention comprises a serum-type 23A capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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.

[0424] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 23A glycoconjugate of the present invention is between 500 kDa and 10,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 23A glycoconjugate is between 1,000 kDa and 7,500 kDa. Preferably, the weight-average molecular weight (Mw) of the serum-type 23A glycoconjugate is between 2,000 kDa and 5,000 kDa.

[0425] The serotype 23A glycoconjugate of the present invention can also be characterized by the ratio (w / w) of sugar to carrier protein. In some embodiments, the ratio (w / w) of serotype 23A polysaccharide to carrier protein in the glycoconjugate is between 0.5 and 3.0. Preferably, the sugar to carrier protein ratio (w / w) is between 0.7 and 1.5. Even more preferably, the sugar to carrier protein ratio (w / w) is between 0.8 and 1.4.

[0426] Another way to characterize the serum-type 23A glycoconjugate of the present invention is by carrier protein (e.g., CRM). 197 The number of lysine residues conjugated to the sugar in the carrier protein (DT or TT) can be characterized as the range of conjugated lysine residues (degree of conjugation). Evidence of lysine modification of the carrier protein (attributed to covalent linkage with the polysaccharide) can be obtained by amino acid analysis and using conventional methods known to those skilled in the art. Conjugation results in a reduction in the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugated material. In a preferred embodiment, the degree of conjugation of the serotype 23A glycoconjugate of the present invention is between 2 and 20. Preferably, the degree of conjugation of the serotype 23A glycoconjugate of the present invention is between 5 and 15.

[0427] The serotype 23A glycoconjugate and immunogenic composition of the present invention may contain free sugars that are not covalently conjugated with the carrier protein but are still present in the glycoconjugate composition. The free sugars may non-covalently associate with the glycoconjugate (i.e., non-covalently bind to the glycoconjugate, adsorb to the glycoconjugate, or be coated in or by the glycoconjugate).

[0428] In one embodiment, the serotype 23A glycoconjugate comprises less than about 40% free serotype 23A polysaccharide compared 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 compared to the total amount of serotype 23A polysaccharide.

[0429] Serum-type 23A glycoconjugates can also be categorized by their molecular size distribution (K). dCharacterization. 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-feed column to obtain a profile of the molecular size distribution of the conjugates. Large molecules excluded from the pores of the medium elute faster than small molecules. A fractionating collector is used to collect the column eluent. The eluted fractions are colorimetrically analyzed by sugar analysis. To determine K... d The column was calibrated to establish the fraction of molecules with complete size exclusion (V0), (K d =0); and indicates the maximum retention (V) i ), (K d =1). The score that achieves the specified sample attribute (V) e ) through expression K d = (V e - V0) / (V i - V0) and K d Related.

[0430] In one embodiment, in the CL-4B column, at least 40% of the serotype 23A glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 50% of the glycoconjugates have a Kc value less than or equal to 0.3. d In a preferred embodiment, between 50% and 90% of the said serotype 23A glycoconjugate is in K+ on a CL-4B column. d Less than or equal to 0.3.

[0431] Serum-type 23A glycoconjugates are also characterized by maintaining the amount of branched-chain rhamnose residues in the 23A polysaccharide. As mentioned above, it has been found that serum-type 23A polysaccharides can lose branched-chain rhamnose residues (see...). Figure 1 ).

[0432] Therefore, in one embodiment, the Streptococcus pneumoniae serotype 23A glycoconjugate of the present invention comprises Streptococcus pneumoniae serotype 23A capsular polysaccharide, wherein the Streptococcus pneumoniae serotype 23A capsular polysaccharide has a branched-chain rhamnose content greater than 75% compared to the natural Streptococcus pneumoniae serotype 23A capsular polysaccharide, wherein the branched-chain rhamnose content in the natural Streptococcus pneumoniae serotype 23A capsular polysaccharide is considered to be about 100%. In one embodiment, the Streptococcus pneumoniae serotype 23A glycoconjugate of the present invention comprises Streptococcus pneumoniae serotype 23A capsular polysaccharide, wherein the Streptococcus pneumoniae serotype 23A capsular polysaccharide has a branched-chain rhamnose content greater than 90% compared to the natural Streptococcus pneumoniae serotype 23A capsular polysaccharide, wherein the branched-chain rhamnose content in the natural Streptococcus pneumoniae serotype 23A capsular polysaccharide is considered to be about 100%. Preferably, the Streptococcus pneumoniae serotype 23A glycoconjugate of the present invention comprises Streptococcus pneumoniae serotype 23A capsular polysaccharide, wherein the Streptococcus pneumoniae serotype 23A capsular polysaccharide has a branched-chain rhamnose content greater than 95% when compared with the natural Streptococcus pneumoniae serotype 23A capsular polysaccharide, wherein the branched-chain rhamnose content in the natural Streptococcus pneumoniae serotype 23A capsular polysaccharide is considered to be about 100%.

[0433] In a preferred embodiment, the Streptococcus pneumoniae serotype 23A glycoconjugate of the present invention comprises Streptococcus pneumoniae serotype 23A capsular polysaccharide, wherein the Streptococcus pneumoniae serotype 23A capsular polysaccharide has a branched-chain rhamnose content of about 100% compared with the natural Streptococcus pneumoniae serotype 23A capsular polysaccharide, wherein the branched-chain rhamnose content in the natural Streptococcus pneumoniae serotype 23A capsular polysaccharide is considered to be about 100%.

[0434] In one embodiment, the serotype 23A sugar is activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. Subsequently, the activated polysaccharide is directly coupled with a carrier protein (preferably CRM). 197The amino group on the carrier is coupled to the carrier via a spacer group (linker). For example, the spacer group may be cystamine or cysteamine, which produces a thiolated polysaccharide. This thiolated polysaccharide may be coupled to the carrier via a thioether bond obtained after reacting with a maleimide-activated carrier protein (e.g., using N-[γ-maleiminobutyryloxy]succinimide ester (GMBS)) or a halogenated acetylated carrier protein (e.g., using iodoacetamide, N-succinimide bromoacetate (SBA; SIB), N-succinimide (4-iodoacetyl)aminobenzoate (SIAB), sulfosuccinimide (4-iodoacetyl)aminobenzoate (sulfo-SIAB), N-succinimide iodoacetate (SIA), or succinimide 3-[bromoacetylamino]propionate (SBAP)). Preferably, the cyanate is coupled with hexanediamine or adipic hydrazide (ADH), and the amino-derived sugar is coupled to the carrier protein (e.g., CRM) via the carbodiimide (e.g., EDAC or EDC) chemical method through the carboxyl group on the protein carrier. 197 Conjugates. Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348 and WO 96 / 129094.

[0435] Other suitable conjugation techniques use carbodiimide, hydrazine, active ester, norbornene, 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 forming a carbonyl linker by reacting 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 reacting with a protein to form a carbamate bond. This may involve reducing the anolyte 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.

[0436] In a preferred embodiment, the serum-type 23A glycoconjugate of the present invention is prepared using a reductive amination chemical method. According to the present invention, reductive amination comprises two steps: (1) oxidizing (activating) the purified sugar, and (2) reducing the activated sugar and carrier protein to form the glycoconjugate (see, for example, WO2006 / 110381, WO2008 / 079653, WO2008 / 143709, WO2008 / 079732, WO2011 / 110531, WO2012 / 119972, WO2015110941, WO2015110940, WO2018 / 144439, WO2018 / 156491).

[0437] In one embodiment, the serum type 23A glycoconjugate of the present invention is prepared by a method comprising the following steps: conjugating isolated serum type 23A capsular polysaccharide with a carrier protein. (a) reacting the isolated serum type 23A capsular polysaccharide with an oxidizing agent; (b) mixing the activated polysaccharide from step (a) with a carrier protein; and (c) reacting the mixed activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate.

[0438] As mentioned above, the size of the isolated serum-type 23A capsular polysaccharide can be set to a target molecular weight (MW) range prior to oxidation. Therefore, in one embodiment, the isolated serum-type 23A capsular polysaccharide is size-set prior to oxidation.

[0439] In a preferred embodiment, the size of the isolated serum type 23A capsular polysaccharide is reduced by mechanical homogenization. In another embodiment, the size of the isolated serum type 23A polysaccharide is reduced by high-pressure homogenization. In a most preferred embodiment, the size of the isolated serum type 23A capsular polysaccharide is not determined by acid hydrolysis.

[0440] In one embodiment, the isolated serum type 23A capsular polysaccharide was not size-set prior to oxidation.

[0441] In one embodiment, oxidation step (a) is carried out at a pH between 4.5 and 6.5. Preferably, oxidation step (a) is carried out at a pH between 5.0 and 6.0.

[0442] In one embodiment, the oxidation step (a) is carried out at a pH of about 5.0.

[0443] After oxidation step (a), the sugar is called activated and referred to as "activated polysaccharide".

[0444] In one embodiment, the activated serum-type 23A polysaccharide of the present invention has a weight-average molecular weight (Mw) between 50 kDa and 400 kDa. In another 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.

[0445] In one embodiment, the activated serotype 23A polysaccharide of the present invention retains at least 80% of branched-chain rhamnose. In one embodiment, the activated serotype 23A polysaccharide of the present invention retains at least 85% of branched-chain rhamnose. In one embodiment, the activated serotype 23A polysaccharide of the present invention retains at least 90% of branched-chain rhamnose. In a preferred embodiment, the activated serotype 23A polysaccharide of the present invention retains at least 95% of branched-chain rhamnose.

[0446] In a preferred embodiment, the activated serum-type 23A polysaccharide of the present invention retains at least 96% of branched rhamnose.

[0447] In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes the terminal hydroxyl group to an aldehyde. In one embodiment, the oxidizing agent is periodate. For the purposes of this invention, the term "periodate" includes periodate and periodic acid; the term also includes metaperiodate (IO). 4- ) and periodate (IO6) 5- And salts including various periodic acids (such as sodium periodate and potassium periodate).

[0448] In a preferred embodiment, the oxidant is sodium periodate. In another embodiment, the periodate used for oxidation is metaperiodate. In a most preferred embodiment, the periodate used for oxidation is sodium metaperiodate.

[0449] When polysaccharides react with periodate, periodate oxidizes the ortho-hydroxyl group to form a carbonyl or aldehyde group, and causes C / C bond cleavage. For this reason, the term "reacting polysaccharides with periodate" includes the oxidation of the ortho-hydroxyl group by periodate.

[0450] In one embodiment, 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.

[0451] In one embodiment, the degree of oxidation of the activated serum-type 23A polysaccharide (also referred to as the "degree of activation" in this invention document) is between 2 and 20. In a preferred embodiment, the degree of oxidation of the activated serum-type 23A polysaccharide is between 2 and 8. In a most preferred embodiment, the degree of oxidation of the activated serum-type 23A polysaccharide is 5 ± 2.5.

[0452] In one embodiment, the activated serotype 23A polysaccharide and carrier protein are lyophilized before step b). Preferably, lyophilization is performed after step a). In one embodiment, the activated polysaccharide is lyophilized after step a), and the carrier protein is also lyophilized.

[0453] In one embodiment, the activated serum type 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.

[0454] In one embodiment, the activated serotype 23A polysaccharide and the carrier protein are lyophilized independently (discrete lyophilization). In another embodiment, the activated serotype 23A polysaccharide and the carrier protein are lyophilized together (co-lyophilization).

[0455] In one embodiment, freeze-drying occurs in the presence of non-reducing sugars, which may include sucrose, trehalose, raffinose, stachyose, melitriose, dextran, mannitol, lactitol, and isomaltitol. In one embodiment, the sugar is selected from the group consisting of sucrose, trehalose, and mannitol. In one embodiment, the sugar is sucrose, trehalose, or mannitol. In one embodiment, the sugar is sucrose.

[0456] In one embodiment, the initial input ratio (by weight) of the activated serotype 23A capsular polysaccharide to the carrier protein at step b) is between 2:1 and 0.5:1. In another embodiment, the initial input ratio (by weight) of the activated serotype 23A capsular polysaccharide to the carrier protein is between 1.2:1 and 0.6:1. Preferably, the initial input ratio (by weight) of the activated serotype 23A capsular polysaccharide to the carrier protein is between 0.9:1 and 0.7:1.

[0457] 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.

[0458] 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).

[0459] In one embodiment, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent.

[0460] Most preferably, the reduction reaction (c) is carried out in DMSO (dimethyl sulfoxide) solvent.

[0461] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride or zinc borohydride in the presence of Bronsted acid or Lewis acid, or amine boranes such as pyridineborane, 2-methylpyridineborane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3, or 5-ethyl-2-methylpyridineborane (PEMB). 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.

[0462] In one embodiment, a reducing agent in a amount between 0.2 and 5 molar equivalents is used in step c). Preferably, a reducing agent in a amount between 0.2 and 1.5 molar equivalents is used in step c). Most preferably, a reducing agent in a amount between 0.5 and 1.0 molar equivalents is used in step c).

[0463] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate, which can be capped using a suitable capping agent. In one embodiment, the capping agent is sodium borohydride (NaBH4).

[0464] In one embodiment, end-capping is achieved by mixing the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In another embodiment, end-capping is achieved by mixing the product of step c) with 1 to 3 molar equivalents of sodium borohydride.

[0465] Following conjugation with a carrier protein, the serum-type 23A glycoconjugate can be purified (enriched relative to the amount of the glycoprotein conjugate) using a variety of techniques known to those skilled in the art. Such techniques include dialysis, concentration / percolation, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and deep filtration. Therefore, in one embodiment, the method for generating the serum-type 23A glycoconjugate of the present invention includes a step of purifying the glycoconjugate after its generation.

[0466] 1.7 The pneumococcal serotype 23B glycoconjugate of the present invention In one aspect, the present invention relates to pneumococcal serotype 23B glycoconjugates.

[0467] The structure of the polysaccharide of Streptococcus pneumoniae serotype 23B is known in the art (see, for example, Ravenscroft N et al. (2017) Carbohydrate Res. Vol. 450, pp. 19-29 and WO2019050814). The structure of the capsular polysaccharide of serotype 23A is as follows: →4)-β-D-Glc p -(1→4)-[Gro-(2→P→3)]- β-D-Gal p -(1→4)-β-L-Rha p -(1→).

[0468] In one embodiment, the Streptococcus pneumoniae serotype 23B sugar used in this invention is a synthetic carbohydrate.

[0469] However, in a preferred embodiment, the source of the bacterial polysaccharide according to the invention may be Streptococcus pneumoniae serotype 23B bacterial cells. Bacterial strains that can be used as a source of Streptococcus pneumoniae serotype 23B polysaccharide may be obtained from an established culture collection (such as from a streptococcal reference laboratory (Centers for Disease Control and Prevention, Atlanta, GA USA)) or clinical samples.

[0470] Serotype 23B sugar can be obtained directly from bacteria using isolation procedures known to those skilled in the art. It can also be purchased (e.g., from the United States Type Culture Collection (ATCC, Manassas, VA USA) (e.g., references ATCC 548-X, ATCC 549-X, ATCC 550-X)).

[0471] In cases where serotype 23B sugar is obtained directly from bacteria, the bacterial cells can be grown in a culture medium, preferably in a soybean-based medium. Following fermentation of the bacterial cells producing the pneumococcal serotype 23B capsular polysaccharide, the bacterial cells can be lysed to produce cell lysates. The serotype 23B polysaccharide can then be separated from the cell lysates using purification techniques known in the art, including centrifugation, deep filtration, precipitation, ultrafiltration, treatment with activated carbon, percolation, and / or column chromatography (see, for example, US2006 / 0228380, US2006 / 0228381, WO2008 / 118752, and WO2020170190). The purified serotype 23B capsular polysaccharide can then be used to prepare glycoconjugates.

[0472] The isolated serum type 23B capsular sugar obtained by purifying serum type 23B polysaccharide from Streptococcus pneumoniae lysate and optionally setting the size of the purified polysaccharide can be characterized by various parameters, including, for example, weight-average molecular weight (Mw).

[0473] The molecular weight of polysaccharides can be measured by combining size exclusion chromatography (SEC) with multi-angle laser scattering detectors (MALLS).

[0474] In one preferred embodiment, the isolated serum-type 23B capsular polysaccharide (i.e., purified prior to further processing) has a weight-average molecular weight between 100 kDa and 2500 kDa. In one embodiment, the isolated serum-type 23B capsular polysaccharide has a weight-average molecular weight between 250 kDa and 2000 kDa. In one embodiment, the isolated serum-type 23B capsular polysaccharide has a weight-average molecular weight between 300 kDa and 1000 kDa.

[0475] Consider any integer within any of the above ranges as an embodiment of the present invention.

[0476] To produce serum-type 23B conjugates with favorable filterability, immunogenicity, and / or yield, the size of the polysaccharide is set to a target molecular weight range prior to conjugation with the carrier protein. Advantageously, the purified serum-type 23B polysaccharide has a reduced size while retaining key structural features. Size setting can be performed mechanically or chemically.

[0477] In one embodiment, the size of the purified serum-type 23B polysaccharide is reduced by chemical hydrolysis. Chemical hydrolysis can be carried out using a weak acid (e.g., acetic acid, formic acid, propionic acid). Chemical hydrolysis can also be carried out using diluted strong acids (such as dilute hydrochloric acid, dilute sulfuric acid, dilute phosphoric acid, dilute nitric acid, or dilute perchloric acid).

[0478] However, preferably, the size of the purified serum-type 23B polysaccharide is reduced by mechanical homogenization. In one embodiment, the size of the purified serum-type 23B polysaccharide is reduced by high-pressure homogenization. High-pressure homogenization achieves a high shear rate by pumping the process fluid through a flow path with a sufficiently small size. The shear rate is increased by using a larger applied homogenization pressure, and the exposure time can be increased by recirculating the feed stream through the homogenizer.

[0479] In one embodiment, the isolated serum-type 23B capsular polysaccharide is sized to have a weight-average molecular weight between 50 kDa and 750 kDa. In a preferred embodiment, the isolated serum-type 23B capsular polysaccharide is sized to have a weight-average molecular weight between 75 kDa and 400 kDa. In an even more preferred embodiment, the isolated serum-type 23B capsular polysaccharide is sized to have a weight-average molecular weight between 100 kDa and 250 kDa. Preferably, the isolated serum-type 23B polysaccharide is sized by mechanical homogenization, preferably by high-pressure homogenization.

[0480] In one embodiment, the isolated serum-type 23B capsular polysaccharide was not size-set.

[0481] In one embodiment, the isolated serotype 23B capsular polysaccharide has a weight-average molecular weight between 50 kDa and 750 kDa prior to conjugation. In another embodiment, the isolated serotype 23B capsular polysaccharide has a weight-average molecular weight between 75 kDa and 400 kDa prior to conjugation. In an even more preferred embodiment, the isolated serotype 23B capsular polysaccharide has a weight-average molecular weight between 100 kDa and 250 kDa prior to conjugation.

[0482] The weight-average molecular weight (Mw) of the sugar prior to conjugation refers to the Mw before polysaccharide activation (i.e., after the final size setting step but before the polysaccharide reacts with the activator). In the case of this invention, the Mw of the 23B polysaccharide is substantially unchanged by the activation step, and the Mw of the 23B polysaccharide incorporated into the conjugate is similar to the Mw of the polysaccharide measured prior to activation.

[0483] In one embodiment, the serotype 23B glycoconjugate of the present invention comprises a serotype 23B capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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.

[0484] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 23B glycoconjugate of the present invention is between 250 kDa and 7,500 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 23B glycoconjugate is between 500 kDa and 4,000 kDa. Preferably, the weight-average molecular weight (Mw) of the serum-type 23B glycoconjugate is between 700 kDa and 2,000 kDa.

[0485] The serotype 23B glycoconjugate of the present invention can also be characterized by the ratio (w / w) of sugar to carrier protein. In some embodiments, the ratio (w / w) of serotype 23B polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar to carrier protein ratio (w / w) is between 0.5 and 1.5. Even more preferably, the sugar to carrier protein ratio (w / w) is between 0.6 and 1.3.

[0486] Another way to characterize the serum-type 23B glycoconjugate of the present invention is by carrier protein (e.g., CRM). 197The number of lysine residues conjugated to the sugar in the carrier protein (DT or TT) can be characterized as the range of conjugated lysine residues (degree of conjugation). Evidence of lysine modification of the carrier protein (attributed to covalent linkage with the polysaccharide) can be obtained by amino acid analysis and using conventional methods known to those skilled in the art. Conjugation results in a reduction in the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugated material. In a preferred embodiment, the degree of conjugation of the serotype 23B glycoconjugate of the present invention is between 2 and 15. Preferably, the degree of conjugation of the serotype 23B glycoconjugate of the present invention is between 5 and 12.

[0487] The serum type 23B glycoconjugate and immunogenic composition of the present invention may contain free sugars that are not covalently conjugated with the carrier protein but are still present in the glycoconjugate composition. The free sugars may non-covalently associate with the glycoconjugate (i.e., non-covalently bind to the glycoconjugate, adsorb to the glycoconjugate, or be coated in or by the glycoconjugate).

[0488] In one embodiment, the serotype 23B glycoconjugate comprises less than about 40% free serotype 23B polysaccharide compared 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 compared to the total amount of serotype 23B polysaccharide.

[0489] Serum-type 23B glycoconjugates can also be categorized by their molecular size distribution (K). d Characterization. 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-feed column to obtain a profile of the molecular size distribution of the conjugates. Large molecules excluded from the pores of the medium elute faster than small molecules. A fractionating collector is used to collect the column eluent. The eluent fractions are colorimetrically analyzed by sugar analysis. To determine K... d The column was calibrated to establish the fraction of molecules with complete size exclusion (V0), (K d =0); and indicates the maximum retention (V) i ), (K d =1). The score that achieves the specified sample attribute (V) e ) through expression K d = (V e - V0) / (V i - V0) and K d Related.

[0490] In one embodiment, in the CL-4B column, at least 30% of the serotype 23B glycoconjugates have a Kc of less than or equal to 0.3. dIn a preferred embodiment, in the CL-4B column, at least 35% of the glycoconjugates have a K0.3 value less than or equal to 0.3. d In a preferred embodiment, 40% to 60% of the serotype 23B glycoconjugate is in K+ on a CL-4B column. d Less than or equal to 0.3.

[0491] In one embodiment, the serotype 23B sugar is activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. Subsequently, the activated polysaccharide is directly coupled with a carrier protein (preferably CRM). 197 The amino group on the carrier is coupled to the carrier via a spacer group (linker). For example, the spacer group may be cystamine or cysteamine, which produces a thiolated polysaccharide. This thiolated polysaccharide may be coupled to the carrier via a thioether bond obtained after reacting with a maleimide-activated carrier protein (e.g., using N-[γ-maleiminobutyryloxy]succinimide ester (GMBS)) or a halogenated acetylated carrier protein (e.g., using iodoacetamide, N-succinimide bromoacetate (SBA; SIB), N-succinimide (4-iodoacetyl)aminobenzoate (SIAB), sulfosuccinimide (4-iodoacetyl)aminobenzoate (sulfo-SIAB), N-succinimide iodoacetate (SIA), or succinimide 3-[bromoacetylamino]propionate (SBAP)). Preferably, the cyanate is coupled with hexanediamine or adipic hydrazide (ADH), and the amino-derived sugar is coupled to the carrier protein (e.g., CRM) via the carbodiimide (e.g., EDAC or EDC) chemical method through the carboxyl group on the protein carrier. 197 Conjugates. Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348 and WO 96 / 129094.

[0492] Other suitable conjugation techniques use carbodiimide, hydrazine, active ester, norbornene, 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 forming a carbonyl linker by reacting 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 reacting with a protein to form a carbamate bond. This may involve reducing the anolyte 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.

[0493] In a preferred embodiment, the serum-type 23B glycoconjugate of the present invention is prepared using a reductive amination chemical method. According to the present invention, reductive amination comprises two steps: (1) oxidizing (activating) the purified sugar, and (2) reducing the activated sugar and carrier protein to form the glycoconjugate (see, for example, WO2006 / 110381, WO2008 / 079653, WO2008 / 143709, WO2008 / 079732, WO2011 / 110531, WO2012 / 119972, WO2015110941, WO2015110940, WO2018 / 144439, WO2018 / 156491).

[0494] In one embodiment, the serum type 23B glycoconjugate of the present invention is prepared by a method comprising the following steps: conjugating isolated serum type 23B capsular polysaccharide with a carrier protein. (a) reacting the isolated serum type 23B capsular polysaccharide with an oxidizing agent; (b) mixing the activated polysaccharide from step (a) with a carrier protein; and (c) reacting the mixed activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate.

[0495] As mentioned above, the size of the isolated serum-type 23B capsular polysaccharide can be set to a target molecular weight (MW) range prior to oxidation. Therefore, in one embodiment, the isolated serum-type 23B capsular polysaccharide is size-set prior to oxidation.

[0496] In a preferred embodiment, the size of the isolated serum-type 23B capsular polysaccharide is reduced by mechanical homogenization. In another embodiment, the size of the isolated serum-type 23B polysaccharide is reduced by high-pressure homogenization.

[0497] In one embodiment, the isolated serum-type 23B capsular polysaccharide was not size-set prior to oxidation.

[0498] In one embodiment, oxidation step (a) is carried out at a pH between 4.0 and 6.5. Preferably, oxidation step (a) is carried out at a pH between 4.5 and 5.5.

[0499] In one embodiment, the oxidation step (a) is carried out at a pH of about 5.0.

[0500] After oxidation step (a), the sugar is called activated and referred to as "activated polysaccharide".

[0501] In one embodiment, the activated serum-type 23B polysaccharide of the present invention has a weight-average molecular weight (Mw) between 40 kDa and 600 kDa. In another 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.

[0502] In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes the terminal hydroxyl group to an aldehyde. In one embodiment, the oxidizing agent is periodate. For the purposes of this invention, the term "periodate" includes periodate and periodic acid; the term also includes metaperiodate (IO). 4- ) and periodate (IO6) 5- And salts including various periodic acids (such as sodium periodate and potassium periodate).

[0503] In a preferred embodiment, the oxidant is sodium periodate. In another embodiment, the periodate used for oxidation is metaperiodate. In a most preferred embodiment, the periodate used for oxidation is sodium metaperiodate.

[0504] When polysaccharides react with periodate, periodate oxidizes the ortho-hydroxyl group to form a carbonyl or aldehyde group, and causes C / C bond cleavage. For this reason, the term "reacting polysaccharides with periodate" includes the oxidation of the ortho-hydroxyl group by periodate.

[0505] In one embodiment, 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 equivalents of periodate. Most preferably, step a) comprises reacting the polysaccharide with about 0.2 molar equivalents of periodate.

[0506] In one embodiment, the degree of oxidation of the activated serum-type 23B polysaccharide (also referred to as the "degree of activation" in this invention document) is between 2 and 20. In a preferred embodiment, the degree of oxidation of the activated serum-type 23B polysaccharide is between 4 and 15. In a most preferred embodiment, the degree of oxidation of the activated serum-type 23B polysaccharide is 9 ± 3.

[0507] In one embodiment, the activated serotype 23B polysaccharide and carrier protein are lyophilized before step b). Preferably, lyophilization is performed after step a). In one embodiment, the activated polysaccharide is lyophilized after step a), and the carrier protein is also lyophilized.

[0508] In one embodiment, the activated serum type 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.

[0509] In one embodiment, the activated serotype 23B polysaccharide and carrier protein are lyophilized independently (discrete lyophilization). In another embodiment, the activated serotype 23B polysaccharide and carrier protein are lyophilized together (co-lyophilization).

[0510] In one embodiment, freeze-drying occurs in the presence of non-reducing sugars, which may include sucrose, trehalose, raffinose, stachyose, melitriose, dextran, mannitol, lactitol, and isomaltitol. In one embodiment, the sugar is selected from the group consisting of sucrose, trehalose, and mannitol. In one embodiment, the sugar is sucrose, trehalose, or mannitol. In one embodiment, the sugar is sucrose.

[0511] In one embodiment, the initial input ratio (by weight) of the activated serotype 23B capsular polysaccharide to the carrier protein at step b) is between 2:1 and 0.5:1. In another embodiment, the initial input ratio (by weight) of the activated serotype 23B capsular polysaccharide to the carrier protein is between 1.2:1 and 0.6:1. Preferably, the initial input ratio (by weight) of the activated serotype 23B capsular polysaccharide to the carrier protein is between 0.9:1 and 0.7:1.

[0512] 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.

[0513] 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).

[0514] In one embodiment, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent.

[0515] Most preferably, the reduction reaction (c) is carried out in DMSO (dimethyl sulfoxide) solvent.

[0516] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride or zinc borohydride in the presence of Brønsted acid or Lewis acid, or amine boranes such as pyridineborane, 2-methylpyridineborane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3, or 5-ethyl-2-methylpyridineborane (PEMB). 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.

[0517] In one embodiment, a reducing agent in a amount between 0.2 and 5 molar equivalents is used in step c). Preferably, a reducing agent in a amount between 0.5 and 1.5 molar equivalents is used in step c). Most preferably, a reducing agent in a amount between 0.9 and 1.1 molar equivalents is used in step c).

[0518] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate, which can be capped using a suitable capping agent. In one embodiment, the capping agent is sodium borohydride (NaBH4).

[0519] In one embodiment, end-capping is achieved by mixing the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In another embodiment, end-capping is achieved by mixing the product of step c) with 1 to 3 molar equivalents of sodium borohydride.

[0520] Following conjugation with a carrier protein, the serum-type 23B glycoconjugate can be purified (enriched relative to the amount of the glycoprotein conjugate) using a variety of techniques known to those skilled in the art. Such techniques include dialysis, concentration / percolation, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and deep filtration. Therefore, in one embodiment, the method for generating the serum-type 23B glycoconjugate of the present invention includes a step of purifying the glycoconjugate after its generation.

[0521] 1.8 The pneumococcal serotype 24F glycoconjugate of the present invention In one aspect, the present invention relates to Streptococcus pneumoniae serotype 24F glycoconjugates.

[0522] The structure of the 24F polysaccharide of Streptococcus pneumoniae serotype is known in the art (see, for example, WO2019050815).

[0523] In one embodiment, the Streptococcus pneumoniae serotype 24F sugar used in this invention is a synthetic carbohydrate.

[0524] However, in a preferred embodiment, the source of the bacterial polysaccharide according to the invention may be Streptococcus pneumoniae serotype 24F bacterial cells. Bacterial strains that can be used as a source of Streptococcus pneumoniae serotype 24F polysaccharide may be obtained from an established culture collection (such as from a Streptococcus reference laboratory (Centers for Disease Control and Prevention, Atlanta, GA USA)) or clinical samples.

[0525] Serotype 24F sugar can be obtained directly from bacteria using isolation procedures known to those skilled in the art. It can also be purchased (e.g., from the United States Type Culture Collection (ATCC, Manassas, VA USA) (e.g., references ATCC 551-X, ATCC 552-X, ATCC 553-X)).

[0526] In cases where serotype 24F sugar is obtained directly from bacteria, the bacterial cells can be grown in a culture medium, preferably in a soybean-based medium. Following fermentation of the bacterial cells producing the pneumococcal serotype 24F capsular polysaccharide, the bacterial cells can be lysed to produce cell lysates. The serotype 24F polysaccharide can then be separated from the cell lysates using purification techniques known in the art, including centrifugation, deep filtration, precipitation, ultrafiltration, treatment with activated carbon, percolation, and / or column chromatography (see, for example, US2006 / 0228380, US2006 / 0228381, WO2008 / 118752, and WO2020170190). The purified serotype 24F capsular polysaccharide can then be used to prepare glycoconjugates.

[0527] The isolated serum-type 24F capsular sugar obtained by purifying serum-type 24F polysaccharide from Streptococcus pneumoniae lysate and optionally setting the size of the purified polysaccharide can be characterized by various parameters, including, for example, weight-average molecular weight (Mw).

[0528] The molecular weight of polysaccharides can be measured by combining size exclusion chromatography (SEC) with multi-angle laser scattering detectors (MALLS).

[0529] In one preferred embodiment, the isolated serum-type 24F capsular polysaccharide (i.e., purified prior to further processing) has a weight-average molecular weight between 100 kDa and 2500 kDa. In one embodiment, the isolated serum-type 24F capsular polysaccharide has a weight-average molecular weight between 250 kDa and 2000 kDa. In one embodiment, the isolated serum-type 24F capsular polysaccharide has a weight-average molecular weight between 500 kDa and 1000 kDa.

[0530] Consider any integer within any of the above ranges as an embodiment of the present invention.

[0531] To produce serum-type 24F conjugates with favorable filterability, immunogenicity, and / or yield, the size of the polysaccharide is set to the target molecular weight range prior to conjugation with the carrier protein. Advantageously, the purified serum-type 24F polysaccharide has a reduced size while retaining key structural features. Size setting can be performed mechanically or chemically.

[0532] Most preferably, the size of the purified serum-type 24F polysaccharide is reduced by mechanical homogenization.

[0533] It has been found that the use of acid hydrolysis recommended, for example, in WO2019050815 or WO2019050818, is not suitable for reducing the size of serum-type 24F polysaccharide.

[0534] Acid hydrolysis has been found to affect the structural integrity of serum-type 24F polysaccharide. It has even been found that relatively mild hydrolysis (e.g., treatment with acetic acid at 100 mM and 80°C for approximately 2 hours) can cause a 25% loss of branched-chain ribose residues (see [link to original text]). Figure 2 ).

[0535] Mechanically sized residues are prevented from being lost, which may be important from an immunological point of view.

[0536] Therefore, in a preferred embodiment, the size of the purified serum-type 24F polysaccharide is reduced by mechanical homogenization.

[0537] In one embodiment, the size of the purified serum-type 24F polysaccharide is reduced by high-pressure homogenization. High-pressure homogenization achieves a high shear rate by pumping the process fluid through a flow path with a sufficiently small size. The shear rate is increased by using a larger applied homogenization pressure, and the exposure time can be increased by recirculating the feed stream through the homogenizer.

[0538] In a preferred embodiment, the method for preparing the serum-type 24F glycoconjugate of the present invention does not include the step of size setting of the serum-type 24F polysaccharide by acid hydrolysis.

[0539] In one embodiment, the isolated serum-type 24F capsular polysaccharide is sized to have a weight-average molecular weight between 50 kDa and 500 kDa. In a preferred embodiment, the isolated serum-type 24F capsular polysaccharide is sized to have a weight-average molecular weight between 75 kDa and 400 kDa. In an even more preferred embodiment, the isolated serum-type 24F capsular polysaccharide is sized to have a weight-average molecular weight between 125 kDa and 275 kDa. In a most preferred embodiment, the isolated serum-type 24F capsular polysaccharide is sized to have a weight-average molecular weight between 125 kDa and 225 kDa. Preferably, the isolated serum-type 24F polysaccharide is sized by mechanical homogenization, preferably by high-pressure homogenization.

[0540] In one embodiment, the isolated serum-type 24F capsular polysaccharide was not size-set.

[0541] In one embodiment, the isolated serotype 24F capsular polysaccharide has a weight-average molecular weight between 50 kDa and 500 kDa prior to conjugation. In another embodiment, the isolated serotype 24F capsular polysaccharide has a weight-average molecular weight between 75 kDa and 400 kDa prior to conjugation. In an even more preferred embodiment, the isolated serotype 24F capsular polysaccharide has a weight-average molecular weight between 125 kDa and 275 kDa prior to conjugation. In a most preferred embodiment, the isolated serotype 24F capsular polysaccharide has a weight-average molecular weight between 125 kDa and 225 kDa prior to conjugation.

[0542] The weight-average molecular weight (Mw) of the sugar prior to conjugation refers to the Mw before polysaccharide activation (i.e., after the final size setting step but before the polysaccharide reacts with the activator). In the case of this invention, the Mw of the 24F polysaccharide is substantially unchanged by the activation step, and the Mw of the 24F polysaccharide incorporated into the conjugate is similar to the Mw of the polysaccharide measured prior to activation.

[0543] In one embodiment, the serum-type 24F glycoconjugate of the present invention comprises a serum-type 24F capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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.

[0544] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 24F glycoconjugate of the present invention is between 500 kDa and 10,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 24F glycoconjugate is between 1,500 kDa and 7,500 kDa. Preferably, the weight-average molecular weight (Mw) of the serum-type 24F glycoconjugate is between 3,000 kDa and 6,000 kDa.

[0545] The serotype 24F glycoconjugate of the present invention can also be characterized by the ratio (w / w) of sugar to carrier protein. In some embodiments, the ratio (w / w) of serotype 24F polysaccharide to carrier protein in the glycoconjugate is between 0.5 and 3.0. Preferably, the sugar to carrier protein ratio (w / w) is between 0.7 and 1.5. Even more preferably, the sugar to carrier protein ratio (w / w) is between 0.8 and 1.4.

[0546] Another way to characterize the serum-type 24F glycoconjugate of the present invention is by carrier protein (e.g., CRM). 197 The number of lysine residues conjugated to the sugar in the carrier protein (DT or TT) can be characterized as the range of conjugated lysine residues (degree of conjugation). Evidence of lysine modification of the carrier protein (attributed to covalent linkage with the polysaccharide) can be obtained by amino acid analysis and using conventional methods known to those skilled in the art. Conjugation results in a reduction in the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugated substance. In a preferred embodiment, the degree of conjugation of the serum-type 24F glycoconjugate of the present invention is between 2 and 15. Preferably, the degree of conjugation of the serum-type 24F glycoconjugate of the present invention is between 5 and 12.

[0547] The serum-type 24F glycoconjugate and immunogenic composition of the present invention may contain free sugars that are not covalently conjugated with the carrier protein but are still present in the glycoconjugate composition. The free sugars may non-covalently associate with the glycoconjugate (i.e., non-covalently bind to the glycoconjugate, adsorb to the glycoconjugate, or be coated in or by the glycoconjugate).

[0548] In one embodiment, the serotype 24F glycoconjugate comprises less than about 40% free serotype 24F polysaccharide compared 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 compared to the total amount of serotype 24F polysaccharide.

[0549] Serum-type 24F glycoconjugates can also be categorized by their molecular size distribution (K). dCharacterization. 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-feed column to obtain a profile of the molecular size distribution of the conjugates. Large molecules excluded from the pores of the medium elute faster than small molecules. A fractionating collector is used to collect the column eluent. The eluent fractions are colorimetrically analyzed by sugar analysis. To determine K... d The column was calibrated to establish the fraction of molecules with complete size exclusion (V0), (K d =0); and indicates the maximum retention (V) i ), (K d =1). The score that achieves the specified sample attribute (V) e ) through expression K d = (V e - V0) / (V i - V0) and K d Related.

[0550] In one embodiment, in the CL-4B column, at least 40% of the serotype 24F glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 50% of the glycoconjugates have a Kc value less than or equal to 0.3. d In a preferred embodiment, between 50% and 90% of the said serotype 24F glycoconjugate is in K+ on a CL-4B column. d Less than or equal to 0.3.

[0551] The serum-type 24F glycoconjugate is also characterized by the amount of branched-chain ribose residues maintained in the 24F polysaccharide. As mentioned above, it has been found that serum-type 24F polysaccharides can lose branched-chain ribose residues (see Figure 7).

[0552] Therefore, in one embodiment, the *Streptococcus pneumoniae* serotype 24F glycoconjugate of the present invention comprises *Streptococcus pneumoniae* serotype 24F capsular polysaccharide, wherein the *Streptococcus pneumoniae* serotype 24F capsular polysaccharide has a ribose content greater than 75% compared to the natural *Streptococcus pneumoniae* serotype 24F capsular polysaccharide, wherein the ribose content in the natural *Streptococcus pneumoniae* serotype 24F capsular polysaccharide is considered to be about 100%. In one embodiment, the *Streptococcus pneumoniae* serotype 24F glycoconjugate of the present invention comprises *Streptococcus pneumoniae* serotype 24F capsular polysaccharide, wherein the *Streptococcus pneumoniae* serotype 24F capsular polysaccharide has a ribose content greater than 90% compared to the natural *Streptococcus pneumoniae* serotype 24F capsular polysaccharide, wherein the ribose content in the natural *Streptococcus pneumoniae* serotype 24F capsular polysaccharide is considered to be about 100%. Preferably, the Streptococcus pneumoniae serotype 24F glycoconjugate of the present invention comprises Streptococcus pneumoniae serotype 24F capsular polysaccharide, wherein the Streptococcus pneumoniae serotype 24F capsular polysaccharide has a ribose content greater than 95% when compared with the natural Streptococcus pneumoniae serotype 24F capsular polysaccharide, wherein the ribose content in the natural Streptococcus pneumoniae serotype 24F capsular polysaccharide is considered to be about 100%.

[0553] In one preferred embodiment, the Streptococcus pneumoniae serotype 24F glycoconjugate of the present invention comprises Streptococcus pneumoniae serotype 24F capsular polysaccharide, wherein the Streptococcus pneumoniae serotype 24F capsular polysaccharide has about 100% ribose content compared to the natural Streptococcus pneumoniae serotype 24F capsular polysaccharide, wherein the ribose content in the natural Streptococcus pneumoniae serotype 24F capsular polysaccharide is considered to be about 100%.

[0554] In one embodiment, the serum-type 24F sugar is activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. Subsequently, the activated polysaccharide is directly coupled with a carrier protein (preferably CRM). 197The amino group on the carrier is coupled to the carrier via a spacer group (linker). For example, the spacer group may be cystamine or cysteamine, which produces a thiolated polysaccharide. This thiolated polysaccharide may be coupled to the carrier via a thioether bond obtained after reacting with a maleimide-activated carrier protein (e.g., using N-[γ-maleiminobutyryloxy]succinimide ester (GMBS)) or a halogenated acetylated carrier protein (e.g., using iodoacetamide, N-succinimide bromoacetate (SBA; SIB), N-succinimide (4-iodoacetyl)aminobenzoate (SIAB), sulfosuccinimide (4-iodoacetyl)aminobenzoate (sulfo-SIAB), N-succinimide iodoacetate (SIA), or succinimide 3-[bromoacetylamino]propionate (SBAP)). Preferably, the cyanate is coupled with hexanediamine or adipic hydrazide (ADH), and the amino-derived sugar is coupled to the carrier protein (e.g., CRM) via the carbodiimide (e.g., EDAC or EDC) chemical method through the carboxyl group on the protein carrier. 197 Conjugates. Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348 and WO 96 / 129094.

[0555] Other suitable conjugation techniques use carbodiimide, hydrazine, active ester, norbornene, 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 forming a carbonyl linker by reacting 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 reacting with a protein to form a carbamate bond. This may involve reducing the anolyte 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.

[0556] In a preferred embodiment, the serum-type 24F glycoconjugate of the present invention is prepared using a reductive amination chemical method. According to the present invention, reductive amination comprises two steps: (1) oxidizing (activating) the purified sugar, and (2) reducing the activated sugar and carrier protein to form the glycoconjugate.

[0557] In one embodiment, the serum-type 24F glycoconjugate of the present invention is prepared by conjugating isolated serum-type 24F capsular polysaccharide with a carrier protein by a method comprising the following steps: (a) reacting the isolated serum-type 24F capsular polysaccharide with an oxidizing agent; (b) mixing the activated polysaccharide from step (a) with the carrier protein; and (c) reacting the mixed activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate.

[0558] As mentioned above, the size of the isolated serum-type 24F capsular polysaccharide can be set to a target molecular weight (MW) range prior to oxidation. Therefore, in one embodiment, the isolated serum-type 24F capsular polysaccharide is size-set prior to oxidation.

[0559] In a preferred embodiment, the size of the isolated serum-type 24F capsular polysaccharide is reduced by mechanical homogenization. In another embodiment, the size of the isolated serum-type 24F polysaccharide is reduced by high-pressure homogenization. In a most preferred embodiment, the size of the isolated serum-type 24F capsular polysaccharide is not determined by acid hydrolysis.

[0560] In one embodiment, the isolated serum-type 24F capsular polysaccharide was not size-set prior to oxidation.

[0561] In one embodiment, oxidation step (a) is carried out at a pH between 4.5 and 6.5. Preferably, oxidation step (a) is carried out at a pH between 5.0 and 6.0.

[0562] In one embodiment, the oxidation step (a) is carried out at a pH of about 5.0.

[0563] After oxidation step (a), the sugar is called activated and referred to as "activated polysaccharide".

[0564] In one embodiment, the activated serum-type 24F polysaccharide of the present invention has a weight-average molecular weight (Mw) between 50 kDa and 400 kDa. In another 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.

[0565] In one embodiment, the activated serum-type 24F polysaccharide of the present invention retains at least 75% branched-chain ribose. In another embodiment, the activated serum-type 24F polysaccharide of the present invention retains at least 90% branched-chain ribose. In a preferred embodiment, the activated serum-type 24F polysaccharide of the present invention retains at least 95% branched-chain ribose.

[0566] In one preferred embodiment, the activated serum-type 24F polysaccharide of the present invention retains approximately 100% of branched-chain ribose.

[0567] In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes the terminal hydroxyl group to an aldehyde. In one embodiment, the oxidizing agent is periodate. For the purposes of this invention, the term "periodate" includes periodate and periodic acid; the term also includes metaperiodate (IO). 4- ) and periodate (IO6) 5- And salts including various periodic acids (such as sodium periodate and potassium periodate).

[0568] In a preferred embodiment, the oxidant is sodium periodate. In another embodiment, the periodate used for oxidation is metaperiodate. In a most preferred embodiment, the periodate used for oxidation is sodium metaperiodate.

[0569] When polysaccharides react with periodate, periodate oxidizes the ortho-hydroxyl group to form a carbonyl or aldehyde group, and causes C / C bond cleavage. For this reason, the term "reacting polysaccharides with periodate" includes the oxidation of the ortho-hydroxyl group by periodate.

[0570] In one embodiment, 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.

[0571] In one embodiment, the degree of oxidation of the activated serum-type 24F polysaccharide (also referred to as the "degree of activation" in this invention document) is between 2 and 20. In a preferred embodiment, the degree of oxidation of the activated serum-type 24F polysaccharide is between 4 and 15. In a most preferred embodiment, the degree of oxidation of the activated serum-type 24F polysaccharide is 9 ± 3.

[0572] In one embodiment, the activated serum-type 24F polysaccharide and carrier protein are lyophilized before step b). Preferably, lyophilization is performed after step a). In one embodiment, the activated polysaccharide is lyophilized after step a), and the carrier protein is also lyophilized.

[0573] In one embodiment, the activated serum-type 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.

[0574] In one embodiment, the activated serotype 24F polysaccharide and the carrier protein are lyophilized independently (discrete lyophilization). In another embodiment, the activated serotype 24F polysaccharide and the carrier protein are lyophilized together (co-lyophilization).

[0575] In one embodiment, freeze-drying occurs in the presence of non-reducing sugars, which may include sucrose, trehalose, raffinose, stachyose, melitriose, dextran, mannitol, lactitol, and isomaltitol. In one embodiment, the sugar is selected from the group consisting of sucrose, trehalose, and mannitol. In one embodiment, the sugar is sucrose, trehalose, or mannitol. In one embodiment, the sugar is sucrose.

[0576] In one embodiment, the initial input ratio (by weight) of the activated serotype 24F capsular polysaccharide to the carrier protein at step b) is between 2:1 and 0.5:1. In another embodiment, the initial input ratio (by weight) of the activated serotype 24F capsular polysaccharide to the carrier protein is between 1.2:1 and 0.6:1. Preferably, the initial input ratio (by weight) of the activated serotype 24F capsular polysaccharide to the carrier protein is between 0.9:1 and 0.7:1.

[0577] 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.

[0578] 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).

[0579] In one embodiment, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent.

[0580] Most preferably, the reduction reaction (c) is carried out in DMSO (dimethyl sulfoxide) solvent.

[0581] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride or zinc borohydride in the presence of Bronsted acid or Lewis acid, or amine boranes such as pyridineborane, 2-methylpyridineborane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3, or 5-ethyl-2-methylpyridineborane (PEMB). 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.

[0582] In one embodiment, a reducing agent in a amount between 0.2 and 5 molar equivalents is used in step c). Preferably, a reducing agent in a amount between 0.5 and 2.5 molar equivalents is used in step c). Most preferably, a reducing agent in a amount between 1.5 and 2.5 molar equivalents is used in step c).

[0583] In one implementation, approximately 2 molar equivalents of reducing agent are used in step c).

[0584] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate, which can be capped using a suitable capping agent. In one embodiment, the capping agent is sodium borohydride (NaBH4).

[0585] In one embodiment, end-capping is achieved by mixing the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In another embodiment, end-capping is achieved by mixing the product of step c) with 1 to 3 molar equivalents of sodium borohydride.

[0586] In one embodiment, capping is achieved by mixing the product of step c) with about 2 molar equivalents of sodium borohydride.

[0587] Following conjugation with a carrier protein, the serum-type 24F glycoconjugate can be purified (enriched relative to the amount of the glycoprotein conjugate) using a variety of techniques known to those skilled in the art. Such techniques include dialysis, concentration / percolation, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and deep filtration. Therefore, in one embodiment, the method for generating the serum-type 24F glycoconjugate of the present invention includes a step of purifying the glycoconjugate after its generation.

[0588] 1.9 The pneumococcal serotype 35B glycoconjugate of the present invention In one aspect, the present invention relates to pneumococcal serotype 23B glycoconjugates.

[0589] The structure of the Streptococcus pneumoniae serotype 35B polysaccharide is known in the art (see, for example, Geno K et al. (2015) Clin Microbiol Rev 28:3, pp. 871-899).

[0590] In one embodiment, the Streptococcus pneumoniae serotype 35B sugar used in this invention is a synthetic carbohydrate.

[0591] However, in a preferred embodiment, the source of the bacterial polysaccharide according to the invention may be Streptococcus pneumoniae serotype 35B bacterial cells. Bacterial strains that can be used as a source of Streptococcus pneumoniae serotype 35B polysaccharide may be obtained from an established culture collection (e.g., from a streptococcal reference laboratory (Centers for Disease Control and Prevention, Atlanta, GA USA)) or clinical samples.

[0592] Serotype 35B sugar can be obtained directly from bacteria using isolation procedures known to those skilled in the art. It can also be purchased (e.g., from the United States Type Culture Collection (ATCC, Manassas, VA USA) (e.g., references ATCC 539-X, ATCC 540-X, ATCC 541-X)).

[0593] In cases where serotype 35B sugar is obtained directly from bacteria, the bacterial cells can be grown in a culture medium, preferably in a soybean-based medium. Following fermentation of the bacterial cells producing the pneumococcal serotype 35B capsular polysaccharide, the bacterial cells can be lysed to produce cell lysates. The serotype 35B polysaccharide can then be separated from the cell lysates using purification techniques known in the art, including centrifugation, deep filtration, precipitation, ultrafiltration, treatment with activated carbon, percolation, and / or column chromatography (see, for example, US2006 / 0228380, US2006 / 0228381, WO2008 / 118752, and WO2020170190). The purified serotype 35B capsular polysaccharide can then be used to prepare glycoconjugates.

[0594] The isolated serum type 35B capsular sugar obtained by purifying serum type 35B polysaccharide from Streptococcus pneumoniae lysate and optionally setting the size of the purified polysaccharide can be characterized by various parameters, including, for example, weight average molecular weight (Mw).

[0595] The molecular weight of polysaccharides can be measured by combining size exclusion chromatography (SEC) with multi-angle laser scattering detectors (MALLS).

[0596] In one preferred embodiment, the isolated serum-type 35B capsular polysaccharide (i.e., purified prior to further processing) has a weight-average molecular weight between 100 kDa and 5000 kDa. In one embodiment, the isolated serum-type 35B capsular polysaccharide has a weight-average molecular weight between 300 kDa and 2000 kDa. In another preferred embodiment, the isolated serum-type 35B capsular polysaccharide has a weight-average molecular weight between 500 kDa and 1000 kDa.

[0597] Consider any integer within any of the above ranges as an embodiment of the present invention.

[0598] The size of purified serum-type 35B polysaccharide can be reduced while retaining key structural features. Size setting can be achieved using either mechanical or chemical methods.

[0599] However, it has been found that Streptococcus pneumoniae serotype 35B polysaccharide cleaves during activation with periodate, a typical oxidizing agent used in commonly used reductive amination methods. It appears that periodate oxidation occurs on the backbone of serotype 35B polysaccharide and cleaves mannitol or ribitol, resulting in a size reduction. Periodate activation causes a decrease in the polysaccharide's molecular weight (Mw). Therefore, serotype 35B capsular polysaccharide is not size-set when activated with periodate.

[0600] Therefore, in one embodiment, the isolated serum-type 35B capsular polysaccharide was not size-set.

[0601] In one embodiment, the isolated serum-type 35B capsular polysaccharide has a weight-average molecular weight between 100 kDa and 5,000 kDa prior to conjugation. In another embodiment, the isolated serum-type 35B capsular polysaccharide has a weight-average molecular weight between 300 kDa and 2,000 kDa prior to conjugation. In an even more preferred embodiment, the isolated serum-type 35B capsular polysaccharide has a weight-average molecular weight between 500 kDa and 1,000 kDa prior to conjugation.

[0602] The weight-average molecular weight (Mw) of a sugar before conjugation refers to the Mw of the polysaccharide before activation (i.e., before the polysaccharide reacts with the activator).

[0603] In one embodiment, the serum-type 35B glycoconjugate of the present invention comprises a serum-type 35B capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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.

[0604] In some embodiments, the weight-average molecular weight (Mw) of the serum-type 35B glycoconjugate of the present invention is between 250 kDa and 7,500 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serum-type 35B glycoconjugate is between 500 kDa and 5,000 kDa. Preferably, the weight-average molecular weight (Mw) of the serum-type 35B glycoconjugate is between 1,000 kDa and 4,000 kDa.

[0605] The serum-type 35B glycoconjugate of the present invention can also be characterized by the ratio (w / w) of sugar to carrier protein. In some embodiments, the ratio (w / w) of serum-type 35B polysaccharide to carrier protein in the glycoconjugate is between 0.4 and 3.0. Preferably, the sugar to carrier protein ratio (w / w) is between 0.4 and 2.0. Even more preferably, the sugar to carrier protein ratio (w / w) is between 0.5 and 1.5.

[0606] Another way to characterize the serum-type 35B glycoconjugate of the present invention is by carrier protein (e.g., CRM). 197 The number of lysine residues conjugated to the sugar in the carrier protein (DT or TT) can be characterized as the range of conjugated lysine residues (degree of conjugation). Evidence of lysine modification of the carrier protein (attributed to covalent linkage with the polysaccharide) can be obtained by amino acid analysis and using conventional methods known to those skilled in the art. Conjugation results in a reduction in the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugated material. In a preferred embodiment, the degree of conjugation of the serum-type 35B glycoconjugate of the present invention is between 2 and 15. Preferably, the degree of conjugation of the serum-type 35B glycoconjugate of the present invention is between 5 and 10.

[0607] The serum-type 35B glycoconjugate and immunogenic composition of the present invention may contain free sugars that are not covalently conjugated with the carrier protein but are still present in the glycoconjugate composition. The free sugars may non-covalently associate with the glycoconjugate (i.e., non-covalently bind to the glycoconjugate, adsorb to the glycoconjugate, or be coated in or by the glycoconjugate).

[0608] In one embodiment, the serotype 35B glycoconjugate comprises less than about 40% free serotype 35B polysaccharide compared to the total amount of serotype 35B polysaccharide. In another embodiment, the serotype 35B glycoconjugate comprises less than about 20% free serotype 35B polysaccharide compared 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 compared to the total amount of serotype 35B polysaccharide.

[0609] Serum-type 35B glycoconjugates can also be categorized by their molecular size distribution (K). d Characterization. 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-feed column to obtain a profile of the molecular size distribution of the conjugates. Large molecules excluded from the pores of the medium elute faster than small molecules. A fractionating collector is used to collect the column eluent. The fractions are colorimetrically analyzed by sugar analysis. To determine K... d The column was calibrated to establish the fraction of molecules with complete size exclusion (V0), (K d=0); and indicates the maximum retention (V) i ), (K d =1). The score that achieves the specified sample attribute (V) e ) through expression K d = (V e - V0) / (V i - V0) and K d Related.

[0610] In one embodiment, in the CL-4B column, at least 40% of the serum-type 35B glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 60% of the glycoconjugates have a K0.3 or less. d In a preferred embodiment, 50% to 80% of the serum-type 35B glycoconjugate is in K+ on a CL-4B column. d Less than or equal to 0.3.

[0611] In one embodiment, the serum-type 35B sugar is activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. Subsequently, the activated polysaccharide is directly coupled with a carrier protein (preferably CRM). 197 The amino group on the carrier is coupled to the carrier via a spacer group (linker). For example, the spacer group may be cystamine or cysteamine, which produces a thiolated polysaccharide. This thiolated polysaccharide may be coupled to the carrier via a thioether bond obtained after reacting with a maleimide-activated carrier protein (e.g., using N-[γ-maleiminobutyryloxy]succinimide ester (GMBS)) or a halogenated acetylated carrier protein (e.g., using iodoacetamide, N-succinimide bromoacetate (SBA; SIB), N-succinimide (4-iodoacetyl)aminobenzoate (SIAB), sulfosuccinimide (4-iodoacetyl)aminobenzoate (sulfo-SIAB), N-succinimide iodoacetate (SIA), or succinimide 3-[bromoacetylamino]propionate (SBAP)). Preferably, the cyanate is coupled with hexanediamine or adipic hydrazide (ADH), and the amino-derived sugar is coupled to the carrier protein (e.g., CRM) via the carbodiimide (e.g., EDAC or EDC) chemical method through the carboxyl group on the protein carrier. 197 Conjugates. Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348 and WO 96 / 129094.

[0612] Other suitable conjugation techniques use carbodiimide, hydrazine, active ester, norbornene, 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 forming a carbonyl linker by reacting 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 reacting with a protein to form a carbamate bond. This may involve reducing the anolyte 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.

[0613] In a preferred embodiment, the serum-type 35B glycoconjugate of the present invention is prepared using a reductive amination chemical method. According to the present invention, reductive amination comprises two steps: (1) oxidizing (activating) the purified sugar, and (2) reducing the activated sugar and carrier protein to form the glycoconjugate.

[0614] In one embodiment, the serum-type 35B glycoconjugate of the present invention is prepared by conjugating isolated serum-type 35B capsular polysaccharide with a carrier protein by comprising the following steps: (a) reacting the isolated serum-type 35B capsular polysaccharide with an oxidizing agent; (b) mixing the activated polysaccharide from step (a) with the carrier protein; and (c) reacting the mixed activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate.

[0615] Preferably, the isolated serum-type 35B capsular polysaccharide was not size-set before oxidation.

[0616] In a preferred embodiment, step (a) is quenched by adding a quenching agent to stop oxidation.

[0617] Therefore, in a preferred embodiment, the serum-type 35B glycoconjugate of the present invention is prepared by conjugating isolated serum-type 35B capsular polysaccharide with a carrier protein by a method comprising the following steps: (a) reacting the isolated serum-type 35B capsular polysaccharide with an oxidizing agent; (a') quenching the oxidation reaction by adding a quenching agent to produce activated serum-type 35B capsular polysaccharide; (b) mixing the activated polysaccharide from step (a') with the carrier protein; and (c) reacting the mixed activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate.

[0618] In one embodiment, oxidation step (a) is carried out at a pH between 5.0 and 7.0. Preferably, oxidation step (a) is carried out at a pH between 5.5 and 6.5.

[0619] In one embodiment, the oxidation step (a) is carried out at a pH of about 6.0.

[0620] After the oxidation steps (a)-(a'), the sugar is called activated and referred to as "activated polysaccharide".

[0621] In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes the terminal hydroxyl group to an aldehyde. In one embodiment, the oxidizing agent is periodate. For the purposes of this invention, the term "periodate" includes periodate and periodic acid; the term also includes metaperiodate (IO). 4- ) and periodate (IO6) 5- And salts including various periodic acids (such as sodium periodate and potassium periodate).

[0622] In a preferred embodiment, the oxidant is sodium periodate. In another embodiment, the periodate used for oxidation is metaperiodate. In a most preferred embodiment, the periodate used for oxidation is sodium metaperiodate.

[0623] When polysaccharides react with periodate, periodate oxidizes the ortho-hydroxyl group to form a carbonyl or aldehyde group, and causes C / C bond cleavage. For this reason, the term "reacting polysaccharides with periodate" includes the oxidation of the ortho-hydroxyl group by periodate.

[0624] In one embodiment, 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.

[0625] In one embodiment, the quencher is selected from vicinal diol, 1,2-aminoethanol, amino acids, glutathione, sulfites, bisulfites, dithionites, metabisulfites, thiosulfates, phosphites, hypophosphites, or phosphorous acid.

[0626] In one embodiment, the quencher is 1,2-aminoethanol of formula (I): Where R 1 Selected from H, methyl, ethyl, propyl or isopropyl.

[0627] In one embodiment, the quenching agent is selected from sodium sulfite and potassium salt, sodium bisulfite and potassium salt, sodium dithionite and potassium salt, sodium metabisulfite and potassium salt, sodium thiosulfate and potassium salt, sodium phosphite and potassium salt, sodium hypophosphite and potassium salt, or sodium phosphite and potassium salt.

[0628] In one embodiment, the quencher is an amino acid. In such embodiments, the amino acid may be selected from serine, threonine, cysteine, cystine, methionine, proline, hydroxyproline, tryptophan, tyrosine, and histidine.

[0629] In one embodiment, the quenching agent is a sulfite, such as a bisulfite, dithionite, metabisulfite, or thiosulfate.

[0630] In one embodiment, the quencher is a compound containing two ortho-hydroxyl groups (ortho-diols), that is, two hydroxyl groups covalently attached to two adjacent carbon atoms.

[0631] Preferably, the quenching agent is a compound of formula (II): Where R 1 and R 2 Each is independently selected from H, methyl, ethyl, propyl or isopropyl.

[0632] In a preferred embodiment, the quencher is glycerol, ethylene glycol, propylene-1,2-diol, butane-1,2-diol, or butane-2,3-diol or ascorbic acid. In a most preferred embodiment, the quencher is butane-2,3-diol.

[0633] In a preferred embodiment, the isolated serum-type 35B polysaccharide is activated by a method comprising the following steps: (a) React the isolated serum type 35B polysaccharide with periodate; (b) An activated serum-type 35B polysaccharide was generated by quenching the oxidation reaction with butan-2,3-diol.

[0634] Following the oxidation step, the polysaccharide is referred to as activated, and will be called "activated polysaccharide" in the following text.

[0635] In one embodiment, the activated serum-type 35B polysaccharide of the present invention has a weight-average molecular weight (Mw) between 15 kDa and 100 kDa. In another 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.

[0636] In one embodiment, the degree of oxidation of the activated serum-type 35B polysaccharide (also referred to as the "degree of activation" in this invention document) is between 2 and 20. In a preferred embodiment, the degree of oxidation of the activated serum-type 35B polysaccharide is between 4 and 15. In a most preferred embodiment, the degree of oxidation of the activated serum-type 35B polysaccharide is 9 ± 3.

[0637] In one embodiment, the activated serum-type 35B polysaccharide and carrier protein are lyophilized before step b). Preferably, lyophilization is performed after step a). In one embodiment, the activated polysaccharide is lyophilized after step a), and the carrier protein is also lyophilized.

[0638] In one embodiment, the activated serum-type 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.

[0639] In one embodiment, the activated serotype 35B polysaccharide and the carrier protein are lyophilized independently (discrete lyophilization). In another embodiment, the activated serotype 35B polysaccharide and the carrier protein are lyophilized together (co-lyophilization).

[0640] In one embodiment, freeze-drying occurs in the presence of non-reducing sugars, which may include sucrose, trehalose, raffinose, stachyose, melitriose, dextran, mannitol, lactitol, and isomaltitol. In one embodiment, the sugar is selected from the group consisting of sucrose, trehalose, and mannitol. In one embodiment, the sugar is sucrose, trehalose, or mannitol. In one embodiment, the sugar is sucrose.

[0641] In one embodiment, the initial input ratio (by weight) of the activated serotype 35B capsular polysaccharide to the carrier protein at step b) is between 2:1 and 0.5:1. In another embodiment, the initial input ratio (by weight) of the activated serotype 35B capsular polysaccharide to the carrier protein is between 1.2:1 and 0.6:1. Preferably, the initial input ratio (by weight) of the activated serotype 35B capsular polysaccharide to the carrier protein is between 0.9:1 and 0.7:1.

[0642] 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.

[0643] 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).

[0644] In one embodiment, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent.

[0645] Most preferably, the reduction reaction (c) is carried out in DMSO (dimethyl sulfoxide) solvent.

[0646] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride or zinc borohydride in the presence of Bronsted acid or Lewis acid, or amine boranes such as pyridineborane, 2-methylpyridineborane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3, or 5-ethyl-2-methylpyridineborane (PEMB). 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.

[0647] In one embodiment, a reducing agent in a amount between 0.2 and 5 molar equivalents is used in step c). Preferably, a reducing agent in a amount between 0.5 and 1.5 molar equivalents is used in step c). Most preferably, a reducing agent in a amount between 0.9 and 1.1 molar equivalents is used in step c).

[0648] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate, which can be capped using a suitable capping agent. In one embodiment, the capping agent is sodium borohydride (NaBH4).

[0649] In one embodiment, end-capping is achieved by mixing the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In another embodiment, end-capping is achieved by mixing the product of step c) with 1 to 3 molar equivalents of sodium borohydride.

[0650] Following conjugation with a carrier protein, the serum-type 35B glycoconjugate can be purified (enriched relative to the amount of the glycoprotein conjugate) using a variety of techniques known to those skilled in the art. Such techniques include dialysis, concentration / percolation, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and deep filtration. Therefore, in one embodiment, the method for generating the serum-type 35B glycoconjugate of the present invention includes a step of purifying the glycoconjugate after its generation.

[0651] 1.10 The pneumococcal serotype 3-saccharide conjugate of the present invention In one aspect, the present invention relates to compositions comprising a Streptococcus pneumoniae serotype 3-saccharide conjugate.

[0652] The structure of the polysaccharide of Streptococcus pneumoniae serotype 3 is known in the art. The repeating unit of the serotype 3 polysaccharide consists of a single pyranose (Glc) p ) and one glucuronic acid (Glc p A) consists of linear disaccharide units (see, for example, Geno K et al. (2015) Clin Microbiol Rev 28:3, pp. 871-899).

[0653] In one embodiment, the Streptococcus pneumoniae serotype 3 sugar used in this invention is a synthetic carbohydrate. The preparation of the synthetic Streptococcus pneumoniae serotype 3 capsular sugar can be carried out, for example, as disclosed in WO2017178664 or WO2015040140.

[0654] However, in a preferred embodiment, the source of the bacterial polysaccharide according to the invention may be Streptococcus pneumoniae serotype 3 bacterial cells. Bacterial strains that can be used as a source of Streptococcus pneumoniae serotype 3 polysaccharide may be obtained from an established culture collection (such as from a streptococcal reference laboratory (Centers for Disease Control and Prevention, Atlanta, GA USA)) or clinical samples.

[0655] Serotype 3 polysaccharide can be obtained directly from bacteria using isolation methods known to those skilled in the art (see, for example, the methods disclosed in US2006 / 0228380, US2006 / 0228381, US2007 / 0184071, US2007 / 0184072, US2007 / 0231340, US2008 / 0102498, and WO2008 / 118752). It can also be produced using synthetic protocols known to those skilled in the art. It can also be purchased (e.g., from the American Center for Type Culture Collection (ATCC, Manassas, VA USA) (e.g., reference numbers ATCC 172-X or ATCC 33-X)).

[0656] In cases where serotype 3 polysaccharide is obtained directly from bacteria, the bacterial cells can be grown in a culture medium, preferably a soybean-based medium. Following fermentation of the bacterial cells producing Streptococcus pneumoniae serotype 3 capsular polysaccharide, the bacterial cells can be lysed to produce cell lysates. The serotype 3 polysaccharide can then be separated from the cell lysates using purification techniques known in the art, including centrifugation, deep filtration, precipitation, ultrafiltration, treatment with activated carbon, percolation, 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 immunogen conjugates.

[0657] The isolated serotype 3 capsular polysaccharide obtained by purifying serotype 3 polysaccharide from Streptococcus pneumoniae lysate and optionally setting the size of the purified polysaccharide can be characterized by various parameters, including, for example, weight-average molecular weight (Mw).

[0658] The molecular weight of polysaccharides can be measured by combining size exclusion chromatography (SEC) with multi-angle laser scattering detectors (MALLS).

[0659] In one embodiment, the isolated serum-type 3 capsular polysaccharide (i.e., purified prior to further processing) has a weight-average molecular weight between 5 kDa and 5,000 kDa. In another embodiment, the isolated capsular polysaccharide has a weight-average molecular weight between 100 kDa and 4,000 kDa. In a preferred embodiment, the isolated capsular polysaccharide has a weight-average molecular weight between 1,000 kDa and 3,500 kDa.

[0660] Preferably, to produce serotype 3 conjugates with advantageous filterability, immunogenicity, and / or yield, the size of the polysaccharide is set to a target molecular weight range prior to conjugation with the carrier protein. Advantageously, the purified serotype 3 polysaccharide has a reduced size while retaining key structural features. Mechanical or chemical size setting can be employed.

[0661] In one embodiment, the size of the purified serum-type 3 polysaccharide is reduced by chemical hydrolysis. The chemical hydrolysis can be carried out using a weak acid (e.g., acetic acid, formic acid, propionic acid). In one embodiment, the chemical hydrolysis is carried out using formic acid. In one embodiment, the chemical hydrolysis is carried out using propionic acid. In a preferred embodiment, the chemical hydrolysis is carried out using acetic acid. The chemical hydrolysis can also be carried out using diluted strong acids (such as dilute hydrochloric acid, dilute sulfuric acid, dilute phosphoric acid, dilute nitric acid, or dilute perchloric acid). In one embodiment, the chemical hydrolysis is carried out using dilute hydrochloric acid. In one embodiment, the chemical hydrolysis is carried out using dilute sulfuric acid. In one embodiment, the chemical hydrolysis is carried out using dilute phosphoric acid. In one embodiment, the chemical hydrolysis is carried out using dilute nitric acid. In one embodiment, the chemical hydrolysis is carried out using dilute perchloric acid.

[0662] The size of purified serum type 3 polysaccharides can also be reduced by mechanical homogenization. In one embodiment, the size of purified serum type 3 polysaccharides is reduced by high-pressure homogenization. High-pressure homogenization achieves a high shear rate by pumping the process fluid through a flow path with a sufficiently small size. The shear rate is increased by using a larger applied homogenization pressure, and the exposure time can be increased by recirculating the feed stream through the homogenizer. The high-pressure homogenization process is suitable for reducing the size of purified serum type 3 polysaccharides while preserving the structural characteristics of the polysaccharides.

[0663] In one embodiment, the isolated serum-type 3-capsular polysaccharide is configured with a weight-average molecular weight between 5 kDa and 1000 kDa. In another embodiment, the isolated serum-type 3-capsular polysaccharide is configured with a weight-average molecular weight between 50 kDa and 300 kDa. In a preferred embodiment, the isolated serum-type 3-capsular polysaccharide is configured with a weight-average molecular weight between 100 kDa and 300 kDa.

[0664] In one embodiment, the size of the isolated serum-type 3 capsular polysaccharide is set to a weight-average molecular weight between about 200 kDa and about 300 kDa.

[0665] In one embodiment, the size of the isolated serum-type 3 capsular polysaccharide is set to a weight-average molecular weight between about 100 kDa and about 200 kDa.

[0666] In one embodiment, the isolated serum-type 3 capsular polysaccharide was not size-set.

[0667] In one embodiment, the serotype 3-saccharide conjugate of the present invention comprises a serotype 3-capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the 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.

[0668] The weight-average molecular weight (Mw) of the serum type 3 polysaccharide prior to conjugation refers to the Mw of the serum type 3 polysaccharide before activation (i.e., after the final size setting step but before the polysaccharide reacts with the activator). In the case of this invention, the Mw of the serum type 3 polysaccharide is substantially unchanged by the activation step, and the Mw of the serum type 3 polysaccharide incorporated into the conjugate is similar to the Mw of the polysaccharide measured prior to activation.

[0669] In one embodiment, the serotype 3 sugar conjugate of the present invention comprises serotype 3 capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the polysaccharide prior to conjugation is between 100 kDa and 200 kDa.

[0670] In one embodiment, the serotype 3 sugar conjugate of the present invention comprises serotype 3 capsular polysaccharide, wherein the weight-average molecular weight (Mw) of the polysaccharide prior to conjugation is between 200 kDa and 300 kDa.

[0671] In some embodiments, the weight-average molecular weight (Mw) of the serotype 3-saccharide conjugate of the present invention is between 250 kDa and 20,000 kDa. In other embodiments, the weight-average molecular weight (Mw) of the serotype 3-saccharide conjugate is between 500 kDa and 15,000 kDa. In still other embodiments, the weight-average molecular weight (Mw) of the serotype 3-saccharide conjugate is between 500 kDa and 10,000 kDa. Preferably, the weight-average molecular weight (Mw) of the serotype 3-saccharide conjugate is between 500 kDa and 5,000 kDa.

[0672] In one embodiment, the weight-average molecular weight (Mw) of the serum-type 3-saccharide conjugate is between 600 kDa and 3,000 kDa.

[0673] The molecular weight of polysaccharides can be measured by combining size exclusion chromatography (SEC) with multi-angle laser scattering detectors (MALLS).

[0674] Another way to characterize the serum-type 3-saccharide conjugate of the present invention is by carrier protein (e.g., CRM). 197 The number of lysine residues conjugated to the sugar in the carrier protein (or SCP) can be characterized as the range of conjugated lysines (degree of conjugation). Evidence of lysine modification of the carrier protein (attributed to covalent linkage with the polysaccharide) can be obtained by amino acid analysis and using conventional methods known to those skilled in the art. Conjugation results in a reduction in the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugated material. In a preferred embodiment, the degree of conjugation of the serotype 3-saccharide conjugate of the present invention is between 2 and 15.

[0675] In a preferred embodiment, the degree of conjugation of the serum-type 3-saccharide conjugate of the present invention is between 4 and 7. In some such embodiments, the carrier protein is CRM. 197 In other such implementations, the carrier protein is SCP.

[0676] The serotype 3 saccharide conjugates of the present invention can also be characterized by the ratio (w / w) of sugar to carrier protein. In some embodiments, the ratio (w / w) of serotype 3 polysaccharide to carrier protein in the saccharide conjugate is between 0.5 and 3.0. In other embodiments, the ratio (w / w) of sugar to carrier protein is between 0.5 and 1.5. In a preferred embodiment, the ratio of serotype 3 capsular polysaccharide to carrier protein in the conjugate is between 0.9 and 1.1.

[0677] The serum-type 3-saccharide conjugate of the present invention can also be characterized by the number of covalent bonds between the carrier protein and the sugar, which varies with the repeating units of the sugar. In one embodiment, the serum-type 3-saccharide conjugate of the present invention contains at least one covalent bond between the carrier protein and the polysaccharide for every four sugar repeating units. In another embodiment, the covalent linkage between the carrier protein and the polysaccharide occurs at least once for every 10 sugar repeating units of the polysaccharide. In another embodiment, the covalent linkage between the carrier protein and the polysaccharide occurs at least once for every 15 sugar repeating units of the polysaccharide. In another embodiment, the covalent linkage between the carrier protein and the polysaccharide occurs at least once for every 25 sugar repeating units of the polysaccharide. In another embodiment, the covalent linkage between the carrier protein and the polysaccharide occurs at least once for every 50 sugar repeating units of the polysaccharide. In another embodiment, the covalent linkage between the carrier protein and the polysaccharide occurs at least once for every 100 sugar repeating units of the polysaccharide.

[0678] In other embodiments, the serum-type 3-saccharide conjugate of the present invention comprises at least one covalent link between the carrier protein and the polysaccharide for every 5 to 10 sugar repeat units of the polysaccharide.

[0679] In other embodiments, the serum-type 3-saccharide conjugate of the present invention comprises at least one covalent link between the carrier protein and the polysaccharide for every 10 to 20 sugar repeat units of the polysaccharide.

[0680] In some implementations, the carrier protein is CRM. 197 And CRM 197 The covalent bond between the SCP and the polysaccharide occurs at least once in every 4, 10, 15, or 25 sugar repeat units of the polysaccharide. In a common implementation, the carrier protein is an SCP, and the covalent link between the SCP and the polysaccharide occurs at least once in every 4, 10, 15, or 25 sugar repeat units of the polysaccharide.

[0681] The serum type 3 glycoconjugate and immunogenic composition of the present invention may contain free sugars that are not covalently conjugated with the carrier protein but are still present in the glycoconjugate composition. The free sugars may non-covalently associate with the glycoconjugate (i.e., non-covalently bind to the glycoconjugate, adsorb to the glycoconjugate, or be coated in or by the glycoconjugate).

[0682] In one preferred embodiment, the serotype 3 saccharide conjugate contains less than about 50% free serotype 3 polysaccharide compared to the total amount of serotype 3 polysaccharide. In another preferred embodiment, the serotype 3 saccharide conjugate contains less than about 40% free serotype 3 polysaccharide compared to the total amount of serotype 3 polysaccharide. In yet another preferred embodiment, the serotype 3 saccharide conjugate contains less than about 25% free serotype 3 polysaccharide compared to the total amount of serotype 3 polysaccharide. In an even more preferred embodiment, the serotype 3 saccharide conjugate contains less than about 20% free serotype 3 polysaccharide compared to the total amount of serotype 3 polysaccharide. In yet another preferred embodiment, the serotype 3 saccharide conjugate contains less than about 15% free serotype 3 polysaccharide compared to the total amount of serotype 3 polysaccharide.

[0683] Serum-type 3-saccharide conjugates can also be categorized by their molecular size distribution (K). d Characterization. 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-feed column to obtain a profile of the molecular size distribution of the conjugates. Large molecules excluded from the pores of the medium elute faster than small molecules. A fractionating collector is used to collect the column eluent. The fractions are colorimetrically analyzed by sugar analysis. To determine K... d The column was calibrated to establish the fraction of molecules with complete size exclusion (V0), (K d =0); and indicates the maximum retention (V) i ), (K d =1). The score that achieves the specified sample attribute (V) e ) through expression K d = (V e - V0) / (V i - V0) and K d Related.

[0684] In a preferred embodiment, in the CL-4B column, at least 30% of the serum-type 3-glycoconjugates have a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 40% of the glycoconjugates have a K0.3 value less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the serum-type 3-glycoconjugate has a Kc of less than or equal to 0.3. d In a preferred embodiment, in the CL-4B column, at least 60% of the serotype 3-glycoconjugates have a Kc value less than or equal to 0.3. d In a preferred embodiment, 50% to 80% of the serotype 3-glycoconjugate is in K+ on a CL-4B column. dLess than or equal to 0.3. In a preferred embodiment, 65% to 80% of the serotype 3-glycoconjugate is in K on a CL-4B column. d Less than or equal to 0.3.

[0685] In one embodiment, the serum-type 3-saccharide conjugate of the present invention is prepared using a reductive amination chemical method (see WO2006110381, WO2008143709, PCT / IB2022 / 054920).

[0686] According to the present invention, reductive amination comprises two steps: (1) oxidizing (activating) the purified sugar, and (2) activating the sugar and carrier protein (e.g., CRM). 197 (TT or SCP) reduction to form glycoconjugates.

[0687] As mentioned above, the size of the polysaccharide can be set to the target molecular weight (MW) range before oxidation.

[0688] Therefore, in one embodiment, the isolated polysaccharide is size-set prior to oxidation.

[0689] In one implementation, the size of the isolated polysaccharide is set to any of the target molecular weight (MW) ranges defined above.

[0690] In one embodiment, the isolated serum type 3 capsular polysaccharide is conjugated to a carrier protein via a method comprising the following steps: (a) React the isolated polysaccharide with an oxidizing agent; (b) Mix the activated polysaccharide from step (a) with the carrier protein; and (c) React the mixed activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate.

[0691] After oxidation step (a), the sugar is called activated and referred to as "activated polysaccharide".

[0692] In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes the terminal hydroxyl group to an aldehyde. In one embodiment, the oxidizing agent is periodate. For the purposes of this invention, the term "periodate" includes periodate and periodic acid; the term also includes metaperiodate (IO). 4- ) and periodate (IO6) 5- ) and salts including various periodic acids (such as sodium periodate and potassium periodate).

[0693] In one embodiment, the oxidant is periodate in the presence of a divalent cation (see WO2008 / 143709).

[0694] 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+ The presence of periodic acid is described. In one embodiment, the oxidant is in Ca... 2+ The presence of periodic acid is a given. In one embodiment, the oxidant is periodate.

[0695] In one embodiment, the oxidant is sodium periodate. In one embodiment, the periodate used for oxidation is metaperiodate. In one embodiment, the periodate used for oxidation is sodium metaperiodate.

[0696] When polysaccharides react with periodate, periodate oxidizes the ortho-hydroxyl group to form a carbonyl or aldehyde group, and causes C / C bond cleavage. For this reason, the term "reacting polysaccharides with periodate" includes the oxidation of the ortho-hydroxyl group by periodate.

[0697] In one embodiment, 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.

[0698] In one embodiment, 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.

[0699] In one preferred embodiment, the degree of oxidation of the activated serum type 3 polysaccharide (also referred to as the "degree of activation" in this invention document) is between 2 and 30. In another preferred embodiment, the degree of oxidation of the activated serum type 3 polysaccharide is between 2 and 20.

[0700] In one embodiment, the degree of oxidation of the activated serum type 3 polysaccharide is between 2 and 8.

[0701] In one embodiment, the degree of oxidation of the activated serum type 3 polysaccharide is between 11 and 19.

[0702] In one embodiment, the activated polysaccharide and carrier protein are lyophilized before step b). Preferably, lyophilization is performed after step a). In one embodiment, the activated polysaccharide is lyophilized after step a), and the carrier protein is also lyophilized.

[0703] In one embodiment, the activated polysaccharide is lyophilized after step a) and the carrier protein is also lyophilized, and the activated polysaccharide and the carrier protein are reconstituted in the same solution, which serves to combine the activated polysaccharide and the carrier protein together.

[0704] In one embodiment, the activated polysaccharide and carrier protein are lyophilized independently (discrete lyophilization). In another embodiment, the activated polysaccharide and carrier protein are lyophilized together (co-lyophilization).

[0705] In one embodiment, freeze-drying occurs in the presence of non-reducing sugars, which may include sucrose, trehalose, raffinose, stachyose, melitriose, dextran, mannitol, lactitol, and isomaltitol. In one embodiment, the sugar is selected from the group consisting of sucrose, trehalose, and mannitol. In one embodiment, the sugar is sucrose, trehalose, or mannitol. In one embodiment, the sugar is trehalose. In one embodiment, the sugar is sucrose.

[0706] In one embodiment, the initial input ratio (by weight) of the activated serotype 3 capsular polysaccharide to the carrier protein at step b) is between 4:1 and 0.1:1. In another embodiment, the initial input ratio (by weight) of the activated serotype 3 capsular polysaccharide to the carrier protein is between 2:1 and 0.4:1.

[0707] In one embodiment, the reduction reaction (c) is carried out in an aqueous solvent.

[0708] In one embodiment, reduction reaction (c) is carried out in an aprotic solvent. In another embodiment, reduction reaction (c) is carried out in DMSO (dimethyl sulfoxide) solvent.

[0709] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride or zinc borohydride in the presence of Bronsted acid or Lewis acid, or amine boranes such as pyridineborane, 2-methylpyridineborane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-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).

[0710] In one embodiment, a reducing agent in step c) is used in an amount between 0.2 and 20 molar equivalents. In another embodiment, a reducing agent in step c) is used in an amount between 0.5 and 3 molar equivalents.

[0711] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate, which can be capped using a suitable capping agent. In one embodiment, the capping agent is sodium borohydride (NaBH4).

[0712] In one embodiment, end-capping is achieved by mixing the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In another embodiment, end-capping is achieved by mixing the product of step c) with 1 to 3 molar equivalents of sodium borohydride.

[0713] In one embodiment, the glycoconjugates of the present invention are prepared using CDI and / or CDT chemical methods (see PCT / IB2022 / 054920).

[0714] The CDI and / or CDT chemical method involves two steps: (1) reacting the isolated sugar with CDI and / or CDT in an aprotic solvent to produce activated sugar (activation), and (2) reacting the activated sugar with a carrier protein (e.g., CRM). 197 (or SCP) reaction to form glycoconjugates.

[0715] In one embodiment, the activator in step (1) is 1,1'-carbonyldiimidazole (CDI). In another embodiment, the activator in step (1) is 1,1'-carbonyl-bis-(1,2,4-triazole) (CDT).

[0716] In one embodiment, the isolated serum type 3 capsular polysaccharide is conjugated to a carrier protein via a method comprising the following steps: (a) React the isolated polysaccharide with CDI and / or CDT in an aprotic solvent; (b) React the activated polysaccharide from step (a) with a carrier protein in an aprotic solvent to form a glycoconjugate.

[0717] In one embodiment, the serum-type 3-saccharide conjugate of the present invention is prepared using a click chemistry method (see, for example, PCT / IB2022 / 054914).

[0718] According to the present invention, the click chemistry method comprises three steps: (a) reacting isolated serum type 3 capsular polysaccharide with a carbonate derivative and an azide linker in an aprotic solvent to produce activated azide polysaccharide (activated polysaccharide); (b) reacting a carrier protein with a reagent containing an N-hydroxysuccinimide (NHS) moiety and an alkyne group, wherein the NHS moiety reacts with an amino group to form an amide bond, thereby obtaining an alkyne-functionalized carrier protein (activation of the carrier protein); and (c) passing through Cu... +1 The mediated azide-alkyne cycloaddition reaction causes the activated azide polysaccharide of step (a) to react with the activated alkyne-carrier protein of step (b) to form a glycoconjugate.

[0719] After step (a), the polysaccharide is referred to as activated and, herein, as “activated polysaccharide” or “activated azidopolysaccharide”.

[0720] After step (b), the carrier is referred to as activated and is called an "activated carrier".

[0721] As mentioned above, the size of the polysaccharide can be set to the target molecular weight (MW) range before activation (a).

[0722] Therefore, in one embodiment, the isolated polysaccharide is size-set before activation with a carbonate derivative and an azide linker.

[0723] In one implementation, the size of the isolated polysaccharide is set to any of the target molecular weight (MW) ranges defined above.

[0724] In one embodiment, the carbonate derivative is 1,1'-carbonyldiimidazole (CDI) or 1,1'-carbonyl-bis-(1,2,4-triazole) (CDT). Preferably, the carbonate derivative is 1,1'-carbonyldiimidazole (CDI).

[0725] In one embodiment, the azide-based linker is a compound of formula (I). Where X is selected from: CH2(CH2) n (CH2CH2O) m CH2CH2, NHCO(CH2) n NHCO(CH2CH2O) m CH2CH2, OCH2(CH2) n and O(CH2CH2O) m CH2CH2; where n is selected from 1 to 10 and m is selected from 1 to 4.

[0726] In one embodiment, the azide-based linker is a compound of formula (II). In one embodiment, the azide-based linker is 3-azidopropylamine.

[0727] In one embodiment, the reagent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is a reagent having an N-hydroxysuccinimide (NHS) moiety and a terminal alkyne.

[0728] In one embodiment, the reagent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is a reagent having an N-hydroxysuccinimide (NHS) moiety and a cycloalkyne.

[0729] In one embodiment, the reagent containing the N-hydroxysuccinimide (NHS) moiety and the alkynyl group is a compound of formula (III). Where X is selected from: CH2O(CH2) n CH2C=O and CH2O(CH2CH2O) m (CH2) n CH2C=O, where n is selected from 0 to 10 and m is selected from 0 to 4.

[0730] In one embodiment, the reagent containing the N-hydroxysuccinimide (NHS) moiety and the alkynyl group is a compound of formula (IV): In one embodiment, step a) comprises reacting the polysaccharide with a carbonate derivative, and then reacting the carbonate-activated polysaccharide with an azide linker in an aprotic solvent to produce an activated azide polysaccharide.

[0731] In one embodiment, at step a), the isolated polysaccharide reacts with a carbonate derivative in an aprotic solvent.

[0732] In a preferred embodiment, the isolated polysaccharide reacts with the carbonate derivative in a solution consisting essentially of dimethyl sulfoxide (DMSO).

[0733] In a preferred embodiment, the isolated polysaccharide is reacted with CDI in dimethyl sulfoxide (DMSO). In another embodiment, the isolated polysaccharide is reacted with CDI in anhydrous DMSO.

[0734] Once the polysaccharide has reacted with the carbonate derivative and the carbonate derivative has been quenched with water, the polysaccharide activated by the carbonate derivative reacts with the azide linker.

[0735] In one embodiment, step a) further comprises reacting the polysaccharide activated by the carbonate derivative with an amount of azide linker in which the amount of polysaccharide repeating units (molar equivalents of RU) relative to the amount of activated polysaccharide is between 0.01 and 10 molar equivalents.

[0736] In one embodiment, the conjugation reaction c) is carried out in an aqueous buffer. In one embodiment, the conjugation reaction c) is carried out in an aqueous buffer in the presence of copper (I) as a catalyst. In one embodiment, the conjugation reaction c) is carried out in an aqueous buffer in the presence of an oxidant and copper (I) as a catalyst. In a preferred embodiment, the conjugation reaction c) is carried out in an aqueous buffer in the presence of copper (I) as a catalyst and ascorbate as an oxidant. In one embodiment, THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) and aminoguanidine may be further added to prevent side reactions of the protein. Therefore, in a preferred embodiment, the conjugation reaction c) is carried out in an aqueous buffer in the presence of copper (I) as a catalyst and ascorbate as an oxidant, wherein the reaction mixture further comprises THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) and aminoguanidine.

[0737] Following the click conjugation reaction, unreacted azide groups may remain in the conjugate, which can be capped using a suitable azide-capping agent. Therefore, in one embodiment, after step c), the unreacted azide groups in the conjugate are capped using a suitable azide-capping agent. In one embodiment, this azide-capping agent is a reagent containing an alkynyl group. In one embodiment, this azide-capping agent is a reagent containing a terminal alkyne. In one embodiment, this azide-capping agent is a reagent containing a cycloalkyne.

[0738] In one embodiment, the azide-based capping agent is a compound of formula (V). Where X is (CH2) n , where n is selected from 1 to 15.

[0739] In one embodiment, the azide-based end-capping agent is propargyl alcohol.

[0740] Therefore, in one embodiment, after step c), the method further includes the step of capping the unreacted azide groups retained in the conjugate with an azide-capping agent.

[0741] After the click conjugation reaction, unreacted alkynyl groups may remain in the conjugate. These unreacted alkynyl groups can be capped using a suitable alkynyl capping agent. In one embodiment, this alkynyl capping agent is a reagent with an azide group.

[0742] In one embodiment, the alkynyl end-capping agent is a compound of formula (VI). Where X is (CH2) n , where n is selected from 1 to 15.

[0743] In one embodiment, the alkynyl end-capping agent is 3-azido-1-propanol.

[0744] Therefore, in one embodiment, after step c), the method further includes the step of capping the unreacted alkynyl groups retained in the conjugate with an alkynyl capping agent.

[0745] Following conjugation with a carrier protein, the glycoconjugate can be purified (enriched in terms of the amount of glyco-protein conjugate) using a variety of techniques known to those skilled in the art. Such techniques include dialysis, concentration / percolation, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and deep filtration. Therefore, in one embodiment, the method for generating the glycoconjugate of the present invention includes a step of purifying the glycoconjugate after its generation.

[0746] In one aspect, the serum-type 3-saccharide conjugate of the present invention is produced according to click chemistry as disclosed above and in patent application PCT / IB2022 / 054914, the entire contents of which are incorporated herein by reference. Thus, in one embodiment, the serum-type 3-saccharide conjugate of the present invention comprises a serum-type 3-saccharide having the general formula (VII) covalently conjugated to a carrier protein (CP) via a spacer: , Where X is selected from: CH2(CH2) n’ (CH2CH2O) m CH2CH2, NHCO(CH2) n’ NHCO(CH2CH2O) m CH2CH2, OCH2(CH2) n’ and O(CH2CH2O) m CH2CH2; where n' is selected from 1 to 10 and m is selected from 1 to 4. And X' is selected from: CH2O(CH2) n’’ CH2C=O, CH2O(CH2CH2O) m’ (CH2) n’’ CH2C=O, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0747] Formula (VII) is a schematic illustration of the serotype 3 sugar conjugate of the present invention. It should not be construed as meaning that there is a linkage on every repeating unit of the sugar. Rather, most of the Streptococcus pneumoniae serotype 3 sugar repeating units remain unmodified, and a few sugar repeating units have covalent bonds between the carrier protein and the sugar. Furthermore, a single carrier protein (CP) molecule can link to more than one Streptococcus pneumoniae serotype 3 sugar molecule, and a single Streptococcus pneumoniae serotype 3 sugar molecule can link to more than one single carrier protein (CP) molecule.

[0748] In a preferred embodiment, the serum-type 3-saccharide conjugate of the present invention comprises a serum-type 3-saccharide 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, where n'' is 1.

[0749] In one embodiment, the serum-type 3-saccharide conjugate of the present invention comprises a serum-type 3-saccharide 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 selected from 1 to 10, and X' is CH2O(CH2). n’’ CH2C=O, where 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 another 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.

[0750] In one embodiment, the serum-type 3-saccharide conjugate of the present invention comprises a serum-type 3-saccharide 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 selected from 1 to 10, and X' is CH2O(CH2CH2O). m’ (CH2) n’’ CH2C=O, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0751] In one embodiment, the serum-type 3-saccharide conjugate of the present invention comprises a serum-type 3-saccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is (CH2CH2O). m CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2). n’’CH2C=O, where 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.

[0752] In one embodiment, the serum-type 3-saccharide conjugate of the present invention comprises a serum-type 3-saccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is (CH2CH2O). m CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2CH2O). m’ (CH2) n’’ CH2C=O, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0753] In one embodiment, the serotype 3-saccharide conjugate of the present invention comprises a serotype 3-saccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is NHCO(CH2). n’ , where n' is selected from 1 to 10, and X' is CH2O(CH2). n’’ CH2C=O, where n'' is selected from 0 to 10.

[0754] In one embodiment, the serotype 3-saccharide conjugate of the present invention comprises a serotype 3-saccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is NHCO(CH2). n’ , where n' is selected from 1 to 10, and X' is CH2O(CH2CH2O). m’ (CH2) n’’ CH2C=O, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0755] In one embodiment, the serum-type 3-saccharide conjugate of the present invention comprises a serum-type 3-saccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is NHCO(CH2CH2O). m CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2). n’’ CH2C=O, where 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.

[0756] In one embodiment, the serum-type 3-saccharide conjugate of the present invention comprises a serum-type 3-saccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is NHCO(CH2CH2O). m CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2CH2O). m’ (CH2) n’’CH2C=O, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0757] In one embodiment, the serotype 3-saccharide conjugate of the present invention comprises a serotype 3-saccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is OCH2(CH2). n’ , where n' is selected from 1 to 10, and X' is CH2O(CH2). n’’ CH2C=O, where n'' is selected from 0 to 10.

[0758] In one embodiment, the serotype 3-saccharide conjugate of the present invention comprises a serotype 3-saccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is OCH2(CH2). n’ , where n' is selected from 1 to 10, and X' is CH2O(CH2CH2O). m’ (CH2) n’’ CH2C=O, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0759] In one embodiment, the serum-type 3-saccharide conjugate of the present invention comprises a serum-type 3-saccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is O (CH2CH2O). m CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2). n’’ CH2C=O, where n'' is selected from 0 to 10.

[0760] In one embodiment, the serum-type 3-saccharide conjugate of the present invention comprises a serum-type 3-saccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII), wherein X is O (CH2CH2O). m CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2CH2O). m’ (CH2) n’’ CH2C=O, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0761] Following conjugation with a carrier protein, the serum-type 3-glycoconjugate can be purified (enriched relative to the amount of the glyco-protein conjugate) using various techniques known to those skilled in the art. Such techniques include dialysis, concentration / percolation, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and deep filtration. Therefore, in one embodiment, the method for producing the glycoconjugate of the present invention includes a step of purifying the glycoconjugate after its production.

[0762] 1.11 Carrier Proteins One component of the glycoconjugate is a carrier protein conjugated to the glycosides of the Streptococcus pneumoniae. The terms "protein carrier," "carrier protein," or "carrier" are used interchangeably herein. The carrier protein should be suitable for standard conjugation methods.

[0763] In a preferred embodiment, the carrier protein of the pneumococcal glycoconjugate is selected from the group consisting of: DT (diphtheria toxoid), TT (tetanus toxoid), or fragments C and CRM of TT. 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 107; and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, ed. Frankel, Maecel Dekker Inc. (1992); Glu-148 deletion or mutation to Asp, Gln, or Ser, and / or Ala 158 deletion or mutation to Gly, and other mutations disclosed in U.S. Patent Nos. 4,709,017 and 4,950,740; mutations of at least one or more residues Lys 516, Lys 526, Phe 530, and / or Lys 534, and other mutations disclosed in U.S. Patent Nos. 5,917,017 and 6,455,673; or fragments disclosed in U.S. Patent No. 5,843,711, Streptococcus pneumoniae pneumococcal hemolysin (ply) (Kuo et al. (1995) Infect lmmun 63:2706-2713) includes some types of antidotes such as dPLY-GMBS (WO 2004 / 081515, WO 2006 / 032499) or dPLY-formol, PhtX (including PhtA, PhtB, PhtD, PhtE (sequences of PhtA, PhtB, PhtD or PhtE are disclosed in WO 00 / 37105 and WO 00 / 39299) and Pht protein fusions such as PhtDE fusions, PhtBE fusions, Pht AE (WO 01 / 98334, WO 03 / 054007, WO2009 / 000826), and OMPC (meningococcal outer membrane protein). (It is usually extracted from Neisseria meningitidis serogroup B (EP0372501)), PorB (from Neisseria meningitidis), PD (Haemophilus influenzae protein D);See, for example, EP0594610 B), or its immune equivalents, synthetic peptides (EP0378881, EP0427347), heat shock proteins (WO 93 / 17712, WO 94 / 03208), pertussis proteins (WO 98 / 58668, EP0471177), cytokines, lymphokines, growth factors or hormones (WO 91 / 01146), artificial proteins containing multiple human CD4+ T cell epitopes derived from various pathogen-derived antigens (Falugi et al. (2001) Eur J Immunol 31:3816-3824), such as N19 protein (Baraldoi et al. (2004) Infect Immun 72:4884-4887), pneumococcal surface protein PspA (WO 02 / 091998), iron uptake protein (WO 01 / 72337), Clostridium difficile toxin A or B (WO 00 / 61761), transferrin-binding protein, Streptococcus pneumoniae adhesion protein (PsaA), recombinant Pseudomonas aeruginosa exotoxin A (especially its non-toxic mutants (e.g., exotoxin A with a substitution at glutamate 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 derivatives of tuberculin (PPD), can also be used as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins, such as cholera toxins (e.g., WO 00 / 61761), 01 / 72337), Clostridium difficile toxin A or B (WO 00 / 61761), transferrin-binding protein, Streptococcus pneumoniae adhesion protein (PsaA), recombinant Pseudomonas aeruginosa exotoxin A (especially its non-toxic mutants (e.g., exotoxin A with a substitution at glutamate 553) (Douglas et al. (1987) J. Bacteriol. 169 (11):4967-4971)). (As described in 2004 / 083251); *Escherichia coli* LT; *Escherichia coli* ST; and exotoxin A from *Pseudomonas aeruginosa*. Another suitable carrier protein is the C5a peptidase (SCP) from Streptococcus. Another suitable carrier protein is rhizavidin [aa 45-179J-GGGGSSS-SP1500-AAA-SP0785] (CP1) (WO2020056202).

[0764] In a preferred embodiment, the carrier protein of the pneumococcal glycoconjugate of the present invention is selected from the group consisting of: TT, DT, DT mutants (e.g., CRM). 197 ) as well as C5a peptidase (SCP) from streptococci.

[0765] In one embodiment, the carrier protein of the pneumococcal glycoconjugate is DT (diphtheria toxoid). In another embodiment, the carrier protein of the pneumococcal glycoconjugate is TT (tetanus toxoid).

[0766] In another embodiment, the carrier protein of the pneumococcal glycoconjugate is PD (Haemophilus influenzae protein D; see, for example, EP0594610 B).

[0767] In a preferred embodiment, the carrier protein of the pneumococcal glycoconjugate is CRM. 197 Or it may be derived from the C5a peptidase (SCP) of Streptococcus.

[0768] In another embodiment, the carrier protein of the pneumococcal glycoconjugate is rhizobium avidin [aa 45-179J-GGGGSSS-SP1500-AAA-SP0785] (CP1).

[0769] In a preferred embodiment, some or all of the pneumococcal glycoconjugate of the present invention is with CRM 197 Protein conjugation. CRM 197 The protein is a non-toxic form of diphtheria toxin, but immunologically indistinguishable from diphtheria toxin. CRM 197 It was caused by the non-toxic bacteriophage β197 tox- Produced by infected Corynebacterium diphtheriae, this avirulent phage is generated by mutagenesis of nitroguanidine in toxin-producing Corynebacterium phage β (Uchida et al. (1971) Nature New Biology 233:8-11). CRM 197 The protein has the same molecular weight as diphtheria toxin, but differs from diphtheria toxin due to a single base change in its structural gene (guanine replaced by adenine). This single base change results in an amino acid substitution in the mature protein (glutamic acid replacing glycine), eliminating the toxic properties of diphtheria toxin. 197 Proteins are safe and effective T-cell-dependent carriers of glycogen. (Regarding CRM) 197 Other details thereof can be found, for example, in U.S. Patent No. 5,614,382.

[0770] In one embodiment, part or all of the pneumococcal glycoconjugate of the present invention is with CRM 197 Protein conjugation. In one embodiment, some or all of the pneumococcal glycoconjugates of the present invention are conjugated with CRM. 197 Protein or CRM 197 The A-chain conjugation (see CN103495161). In one embodiment, part or all of the pneumococcal glycoconjugate of the present invention is with CRM obtained by expression in recombinant Escherichia coli (see CN103495161). 197 A-chain conjugation (see CN103495161).

[0771] In other preferred embodiments, the carrier protein of some or all of the pneumococcal glycoconjugates of the present invention is SCP (streptococcal C5a peptidase). See WO2022249106, particularly pages 95 to 111, which is incorporated herein by reference.

[0772] In one embodiment, the carrier protein of the pneumococcal glycosaminoglycan conjugate of the present invention is SCP ...

Claims

1. An immunogenic composition comprising glycoconjugates from different serotypes of Streptococcus pneumoniae and a saponin-containing liposome adjuvant, said liposome adjuvant comprising a liposome composition containing monophosphoryl lipid A (MPLA) and at least one saponin, wherein said liposome composition comprises: i) a lipid bilayer containing phospholipids and ii) cholesterol.

2. The immunogenic composition of claim 1, wherein the saponin is QS-21.

3. The immunogenic composition according to any one of claims 1 to 2, wherein the phospholipid is DMPC and DMPG.

4. The immunogenic composition of claim 1, wherein the saponin-containing liposome adjuvant comprises monophosphoryl lipid A (MPLA), QS-21, DMPC, DMPG and cholesterol in phosphate buffer.

5. The immunogenic composition according to any one of claims 1 to 4, wherein MPLA is monophosphoryl 3-deacyl lipid A.

6. The immunogenic composition according to any one of claims 1 to 5, comprising about 0.1 to about 1.0 mg / mL or higher of MPLA.

7. The immunogenic composition according to any one of claims 1 to 6, comprising about 0.05 to about 1.0 mg / mL or higher of QS-21.

8. The immunogenic composition according to any one of claims 1 to 7, comprising about 0.5 to about 20 mg / mL or higher of cholesterol.

9. The immunogenic composition according to any one of claims 1 to 8, comprising about 0.5 to about 20 mg / mL or higher of DMPC.

10. The immunogenic composition according to any one of claims 1 to 9, comprising about 0.5 to about 3.0 mg / mL or higher of DMPG.

11. The immunogenic composition of claim 1, wherein the saponin-containing liposome adjuvant comprises about 0.4 mg / mL of monophosphoryl 3-deacyl lipid A, about 0.2 mg / mL of QS-21, about 14 mg / mL of DMPC, about 1.6 mg / mL of DMPG and about 11 mg / mL of cholesterol.

12. The immunogenic composition of claim 1, wherein the saponin-containing liposome adjuvant comprises about 0.8 mg / mL of monophosphoryl 3-deacyl lipid A, about 0.4 mg / mL of QS-21, about 28 mg / mL of DMPC, about 3.2 mg / mL of DMPG and about 22 mg / mL of cholesterol.

13. The immunogenic composition of claim 1, wherein the saponin-containing liposome adjuvant comprises about 0.2 mg / mL of monophosphoryl 3-deacyl lipid A, about 0.1 mg / mL of QS-21, about 7 mg / mL of DMPC, about 0.8 mg / mL of DMPG and about 5.5 mg / mL of cholesterol.

14. The immunogenic composition according to any one of claims 1 to 13, wherein the saponin-containing liposome adjuvant is homogeneous.

15. The immunogenic composition according to any one of claims 1 to 13, wherein the saponin-containing liposome adjuvant is heterogeneous.

16. The immunogenic composition according to any one of claims 1 to 15, comprising at least 20 glycoconjugates derived from different serotypes of Streptococcus pneumoniae.

17. The immunogenic composition according to any one of claims 1 to 16, comprising glycoconjugates 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.

18. The immunogenic composition according to any one of claims 1 to 16, comprising glycoconjugates 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.

19. The immunogenic composition according to any one of claims 1 to 18, comprising glycoconjugates of 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, and additionally comprising at least ten glycoconjugates of Streptococcus pneumoniae serotypes 2, 6C, 7C, 7F, 9N, 10B, 15C, 16F, 17F, 20A, 20B, 21, 22A, 24B, 27, 29, 31, 33B, 34, 35F, 38, 72, and 73.

20. The immunogenic composition of claim 19, wherein it is a 35-valent Streptococcus pneumoniae conjugate composition.

21. The immunogenic composition according to any one of claims 1 to 20, used as a pharmaceutical.

22. The immunogenic composition according to any one of claims 1 to 20, which is used as a vaccine.

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