Media and fermentation methods for polysaccharide production in bacterial cell culture

Abstract

The present disclosure relates to media and fermentation methods for polysaccharide production in bacterial cell culture. In one aspect, the disclosure relates to cell culture media comprising potassium phosphate and calcium chloride. In another aspect, the disclosure relates to a method of fermenting bacterial cell culture under pressurized conditions. The media and methods disclosed herein are useful for increasing the polysaccharide production and reducing the lag phase of a bacterial cell culture, i.e., Streptococcus pneumoniae.

Description

[0001] MEDIA AND FERMENTATION METHODS FOR POLYSACCHARIDE PRODUCTION IN BACTERIAL CELL CULTURE

[0002] BACKGROUND OF THE DISCLOSURE

[0003] Infections caused by pneumococci are a major cause of morbidity and mortality all over the world. Pneumonia, febrile bacteraemia and meningitis are the most common manifestations of invasive pneumococcal disease, whereas bacterial spread within the respiratory tract may result in middle-ear infection, sinusitis or recurrent bronchitis. Compared with invasive disease, the non-invasive manifestations are usually less severe, but considerably more common.

[0004] In Europe and the United States, pneumococcal pneumonia is the most common community-acquired bacterial pneumonia, estimated to affect approximately 100 per 100,000 adults each year. The corresponding figures for febrile bacteraemia and meningitis are 15-19 per 100 000 and 1-2 per 100,000, respectively. The risk for one or more of these manifestations is much higher in infants and elderly people, as well as immune compromised persons of any age. Even in economically developed regions, invasive pneumococcal disease carries high mortality; for adults with pneumococcal pneumonia the mortality rate averages 10%— 20%, whilst it may exceed 50% in the high-risk groups. Pneumonia is by far the most common cause of pneumococcal death worldwide.

[0005] The etiological agent of pneumococcal diseases, Streptococcus pneumoniae (pneumococcus), is a Gram-positive encapsulated coccus, surrounded by a polysaccharide capsule. Differences in the composition of this capsule permit serological differentiation between about 91 capsular types, some of which are frequently associated with pneumococcal disease, others rarely. Invasive pneumococcal infections include pneumonia, meningitis and febrile bacteremia; among the common non-invasive manifestations are otitis media, sinusitis and bronchitis.

[0006] Pneumococcal conjugate vaccines (PCVs) are pneumococcal vaccines used to protect against disease caused by S. pneumoniae (pneumococcus). PCV vaccines available on the global market include Prevnar® (called Prevenar in some countries) (heptavalent vaccine), SYNFLORIX® (a decavalent vaccine), Prevnar 13® (tridecavalent vaccine), Vaxneuvance™ (a 15-valent vaccine), and Prevnar 20™ (a 20-valent vaccine). The recent development of widespread microbial resistance to essential antibiotics and the increasing number of immunocompromised persons underline the need for pneumococcal vaccines with even broader protection.

[0007] In particular, there is a need to address remaining unmet medical need for coverage of pneumococcal disease due to serotypes not found in Prevnar 13® and potential for serotype replacement over time. The specific serotypes causing disease beyond the 13 in Prevnar 13® vary by region, population, and may change over time due to acquisition of antibiotic resistance, pneumococcal vaccine introduction and secular trends of unknown origin. There is a need for immunogenic compositions that can be used to induce an immune response against additional Streptococcus pneumoniae serotypes in humans and in particular in children less than 2 years old.

[0008] Accordingly, there is a need for the development of improved systems for producing Streptococcus pneumoniae polysaccharides by bacterial cell culture.

[0009] SUMMARY

[0010] The present disclosure provides media and methods for producing polysaccharides by a bacterial cell culture. In particular, provided herein are materials and methods to maximize capsular polysaccharide production of bacteria, such as Streptococcus pneumoniae.

[0011] In one aspect, the present disclosure provides a medium comprising potassium phosphate for use in culturing a bacterial cell culture, wherein the bacterial cell culture produces capsular polysaccharides. In some embodiments, the concentration of the potassium phosphate is between about 0.5 grams per liter of medium (g / L) and about 2 g / L. In other embodiments, the concentration of the potassium phosphate is about 1 g / L. In some embodiments, the potassium phosphate is monobasic potassium phosphate.

[0012] In another aspect, the present disclosure provides a medium comprising calcium chloride for use in culturing a bacterial cell culture, wherein the bacterial cell culture produces capsular polysaccharides. In some embodiments, the concentration of the calcium chloride is between about 0.1 g / L and about 0.2 g / L. In other embodiments, the concentration of the calcium chloride is between about 0.13 g / L and about 0.14 g / L. In still other embodiments, the concentration of the calcium chloride is about 0.13374 g / L. In a particular embodiment, the calcium chloride is anhydrous. In other embodiments, the present disclosure provides a medium comprising calcium chloride, potassium phosphate, and glucose for use in culturing a bacterial cell culture, wherein the bacterial cell culture produces capsular polysaccharides. In some embodiments, the glucose has a concentration of about 55 g / L.

[0013] In other embodiments, the present disclosure provides a medium for use in culturing a bacterial cell culture, wherein the medium comprises calcium chloride, potassium phosphate, and a component selected from the list consisting of tyrosine, asparagine, sodium chloride, cysteine, glutamine, and iron sulfate. In still other embodiments, the present disclosure provides a medium for use in culturing a bacterial cell culture, wherein the medium comprises calcium chloride, potassium phosphate, glucose, tyrosine, asparagine, sodium chloride, cysteine, glutamine, and iron sulfate. In some embodiments, the medium comprises calcium chloride, potassium phosphate, and each of the additional components provided in Table 1.

[0014] In another aspect, the present disclosure provides a method of increasing the capsular polysaccharide titer measured in grams per liter (g / L) produced by a bacterial cell culture over a unit of time, comprising culturing the bacterial cell culture in a medium disclosed herein. In some embodiments, the unit of time is about 24 hours. In other embodiments, the polysaccharide titer is at least about 1 g / L or at least about 2 g / L.

[0015] In yet another aspect, the present disclosure provides a method of increasing capsular polysaccharide specific productivity measured in grams polysaccharide per liter per OD (g / L / OD) of a bacterial cell culture over a unit of time, comprising culturing the bacterial cell culture in a medium disclosed herein. In some embodiments, the unit of time is about 24 hours. In some embodiments, the polysaccharide specific productivity is between about 0.1 g / L / OD and about 0.3 g / L / OD.

[0016] In still another aspect, the present disclosure provides a method of culturing a bacterial cell culture in a bioreactor wherein the backpressure is about 1 PSI, about 2 PSI, about 3 PSI, about 4 PSI, or about 5 PSI, and wherein the bacterial cell culture produces capsular polysaccharides. In some embodiments, the backpressure is about 5 PSI. In some embodiments, the lag phase of the bacteria in the bacterial cell culture is decreased as compared to the lag phase of bacteria in a bacterial cell culture cultured in a bioreactor without backpressure. In some embodiments, the lag phase of the bacteria is decreased by between about 0.5 hours and about 5 hours. In further embodiments, the total culturing time required to achieve a target capsular polysaccharide yield is decreased as compared to the total culturing time required to achieve the target capsular polysaccharide yield with a bacterial cell culture cultured in a bioreactor without backpressure. In some embodiments, the total culturing time is decreased by between about 1 hour and about 10 hours. In some embodiments, the target polysaccharide yield is about 1 g / L, about 1 .5 g / L, about 1 .6 g / L, about 1 .7 g / L, about 1 .8 g / L, about 1 .9 g / L, or about 2 g / L.

[0017] In some embodiments, the present disclosure provides a method of culturing a bacterial cell culture in a bioreactor wherein the backpressure is about 1 PSI, about 2 PSI, about 3 PSI, about 4 PSI, or about 5 PSI, wherein the bacterial cell culture produces capsular polysaccharides, and wherein the method does not further comprise the use of nitrogen overlay.

[0018] In some embodiments, the bacterial cell culture used with the media and methods described herein comprises a bacteria selected from the group consisting of Streptococcus agalactiae, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria meningitidis, Escherichia coli, Salmonella typhi, Haemophilus influenzae, Klebsiella pneumoniae, Enterococcus faecium, and Enterococcus faecalis. In a particular embodiment, the bacteria is Streptococcus pneumoniae.

[0019] In particular embodiments, the bacterial cell culture used with the media and methods described herein comprises Streptococcus pneumoniae with a serotype selected from the group consisting of 15A, 23A, 23B, 24F, and 35B.

[0020] In another aspect, provided herein is a capsular polysaccharide produced by the medium or methods described herein. Also described herein is an immunogenic composition comprising a capsular polysaccharide produced by the medium or methods described herein. In some embodiments, the immunogenic composition comprises capsular polysaccharides from at least one of Streptococcus pneumoniae serotypes 15A, 23A, 23B, 24F, and 35B. In other embodiments, the immunogenic composition comprises capsular polysaccharides from each of Streptococcus pneumoniae serotypes 15A, 23A, 23B, 24F, and 35B. In some embodiments, the immunogenic composition comprises capsular polysaccharides from each of Streptococcus pneumoniae serotypes 15A, 23A, 23B, 24F, and 35B, wherein each of the capsular polysaccharides are produced by the medium or methods described herein. In some embodiments, provided herein are immunogenic compositions for use in a method of inducing an immune response to Streptococcus pneumoniae serotypes 15A, 23A, 23B, 24F, and / or 35B. In still other embodiments, the immunogenic composition comprises capsular polysaccharides from each of Streptococcus pneumoniae serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and / or 35B. In some embodiments, provided herein are immunogenic compositions for use in a method of inducing an immune response to Streptococcus pneumoniae serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and / or 35B.

[0021] DETAILED DESCRIPTION

[0022] Before the present composition and methods are described, it is to be understood that this disclosure is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only and is not intended to be limiting. All publications mentioned herein are incorporated by reference in their entirety.

[0023] The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below and throughout the specification.

[0024] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0025] The term "about" or "approximately" means within a statistically meaningful range of a value. Such a range can be within an order of magnitude, typically within 20%, more typically still within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by the term "about" or "approximately" depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art. Whenever a range is recited within this application, every whole number integer within the range is also contemplated as an embodiment of the disclosure.

[0026] The term “batch culture” as used herein refers to a method of culturing cells in which all the components that will ultimately be used in culturing the cells, including the medium as well as the cells themselves, are provided at the beginning of the culturing process. A batch culture is typically stopped at some point and the cells and / or components in the medium are harvested and optionally purified.

[0027] The term “bioreactor” as used herein refers to any vessel used for the growth of a bacterial cell culture. The bioreactor can be of any size so long as it is useful for the culturing of bacterial cells. Typically, the bioreactor will be at least 500 mL and may be 1 ; 10; 50; 75; 100; 250; 500; 1 ,000; 1 ,500; 2,000; 2,500; 5,000; 8,000; 10,000; 12,000; 15,000; 20,000 liters or more, or any volume in between. The internal conditions of the bioreactor, including, but not limited to pH and temperature, are typically controlled during the culturing period. The bioreactor can be composed of any material that is suitable for holding bacterial cell cultures suspended in medium under the culture conditions of the present disclosure, including glass, plastic or metal.

[0028] The term “capsular polysaccharide” or "capsule polysaccharide" refers to a glycopolymer that includes repeating units of one or more monosaccharides joined by glycosidic linkages. A capsular polysaccharide typically forms a capsule-like layer around a bacterial cell.

[0029] The term “cell density” as used herein refers to that number of cells present in a given volume of medium.

[0030] The term “cell viability” as used herein refers to the ability of cells in culture to survive under a given set of culture conditions or experimental variations. The term as used herein also refers to that portion of cells which are alive at a particular time in relation to the total number of cells, living and dead, in the culture at that time.

[0031] The terms “culture”, “cell culture” and “bacterial cell culture” as used herein refer to a bacterial cell population that is suspended in a medium under conditions suitable to survival and / or growth of the cell population. As will be clear to those of ordinary skill in the art, these terms as used herein may refer to the combination comprising the bacterial cell population and the medium in which the population is suspended.

[0032] The term “fed-batch culture” as used herein refers to a method of culturing cells in which additional components are provided to the culture at some time subsequent to the beginning of the culture process. The provided components typically comprise nutritional supplements for the cells which have been depleted during the culturing process. A fed-batch culture is typically stopped at some point and the cells and / or components in the medium are harvested and optionally purified.

[0033] These terms “medium”, “cell culture medium”, “bacterial culture medium”, and “culture medium” as used herein refer to a solution containing nutrients which nourish growing bacterial cells. Typically, these solutions provide essential and non- essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for minimal growth and / or survival. The solution may also contain components that enhance growth and / or survival above the minimal rate, including hormones and growth factors. The solution is typically formulated to a pH and salt concentration optimal for cell survival and proliferation. The medium may also be a “defined medium” - a serum-free medium that contains no proteins, hydrolysates or components of unknown composition. Defined media are free of animal-derived components and all components have a known chemical structure.

[0034] The term “perfusion culture” as used herein refers to a method of culturing cells in which additional components are provided continuously or semi-continuously to the culture subsequent to the beginning of the culture process. The provided components typically comprise nutritional supplements for the cells which have been depleted during the culturing process. A portion of the cells and / or components in the medium, such as metabolic waste products, are typically harvested on a continuous or semi-continuous basis and are optionally purified.

[0035] The term “seeding” as used herein refers to the process of providing a cell culture to a bioreactor or another vessel. The cells may have been propagated previously in another bioreactor or vessel. Alternatively, the cells may have been frozen and thawed immediately prior to providing them to the bioreactor or vessel. The term refers to any number of cells, including a single cell.

[0036] An “oligosaccharide” as used herein refers to a compound containing two or more monosaccharide units or moieties. Within the context of an oligosaccharide, an individual monomer unit or moiety is a monosaccharide which is, or can be, bound through a hydroxyl group to another monosaccharide unit or moiety. Oligosaccharides can be prepared by either chemical synthesis from protected single residue sugars or by chemical degradation of biologically produced polysaccharides. Alternatively, oligosaccharides may be prepared by in vitro enzymatic methods. The term “polysaccharide” (PS) refers to a linear or branched polymer of at least 5 monosaccharide units or moieties. For clarity, larger number of repeating units, wherein n is greater than about 5, such as greater than about 10, will be referred to herein as a polysaccharide.

[0037] The term “titer” as used herein refers to the total amount of polysaccharide produced by a bacterial cell culture divided by a given amount of medium volume. Titer is typically expressed in units of grams of polysaccharide per liter of medium.

[0038] The terms "vaccine" or "vaccine composition", which are used interchangeably, refer to pharmaceutical compositions comprising at least one immunogenic composition that induces an immune response in an animal.

[0039] Bacteria

[0040] Any bacteria having a polysaccharide containing cell wall may be utilized in accordance with the present disclosure. In a particular embodiment, the bacteria are encapsulated bacteria. Non-limiting examples of encapsulated bacteria that may be used in accordance with the present disclosure include Streptococcus species, such as S. agalactiae and S. pneumoniae, Staphylococcus aureus, Neisseria meningitidis, Escherichia coli, Salmonella typhi, Haemophilus influenzae, Klebsiella pneumoniae, Enterococcus faecium, and Enterococcus faecalis. In a some embodiments, the bacteria have fastidious growth requirements. Fastidious bacteria include, but are not limited to, Streptococcus species (e.g. S. agalactiae and S. pneumoniae).

[0041] There are more than 90 different serotypes of S. pneumoniae, any of which are contemplated for use in the present disclosure. Examples include, but are not limited to: 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. For example, in one embodiment, S. pneumoniae serotypes 15A, 23A, 23B, 24F, or 35B may be used in the present disclosure. In another embodiment, serotypes 15A, 23A, 23B, 24F, and 35B may be used in the present disclosure. In still another embodiment, S. pneumoniae serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B may be used in the present disclosure.

[0042] Similarly, any encapsulated strain of S. aureus may be used in the present disclosure. For example, S. aureus strains producing serotype 5 or 8 capsular polysaccharides, such as Reynolds, Becker, Newman, PS80, JL278, and JL812, are contemplated.

[0043] Any strain of / V. meningitidis serogroups A, C, Y, and W-135 may be used in the present disclosure.

[0044] Any strain of E. coli may be used in the present disclosure.

[0045] Any strain of S. typhi X / i may be used in the present disclosure.

[0046] Any strain of H. influenza type b may be used in the present disclosure.

[0047] Any strain of K. pneumoniae may be used in the present disclosure.

[0048] E. faecalis may be used in the present disclosure.

[0049] Additionally, any number of commercially and non-commercially available bacteria having cell wall polysaccharides may be utilized in accordance with the present disclosure. One skilled in the art will appreciate that some bacteria have different nutrition requirements and / or might require different culture conditions for optimal growth and will be able to modify conditions as needed.

[0050] In many instances, the strains of bacteria will be selected or engineered to produce high levels of polysaccharide. In some embodiments, the bacterial cells are genetically engineered to produce high levels of polysaccharide.

[0051] Cell Culture Media

[0052] The present disclosure provides a variety of media formulations that maximize polysaccharide production in bacterial cell cultures.

[0053] Previously described methods for culturing S. pneumoniae have included complex media and defined media (See Sanchez-Rosario Y, et al. Front Cell Infect Microbiol. 2021 Mar 23; 11 :613623). Complex media is prepared using digests of microbial, animal, or plant products. Numerous complex pneumococcal growth media have been described in the art, for example, Todd and Hewitt medium, M16 / M17 medium, and Brain Heart Infusion Broth (See Id. and Wulandari et al. J Microbiol Methods 2022 Sep;200: 106539). These media contain components such as pancreatic digest of casein, yeast extract, beef extract, and rabbit serum.

[0054] Subsequent to the use of complex media, it was discovered that an optimized fully synthetic ( / .e., defined) medium could produce advantageous properties such as increased growth (See Sanchez-Rosario Y, et al. Front Cell Infect Microbiol. 2021 Mar 23; 11 :613623). Numerous defined pneumococcal growth media have been described in the art. For example, Rane and Subarrow, Van de Rijn and Kessler, Sicard, and RPMI 1640 are each media which contain defined amounts of amino acids, vitamins, and inorganic salts (See Id.', Rane, L., and Subbarow, Y. J. Bacterial. 40, 695-704 (1940); Sicard Genetics 50, 31-44 (1964); and Van De Rijn, I., and Kessler, R. E. Infect. Immun. 27 (2), 444-448 (1980)).

[0055] Described herein are methods for culturing polysaccharide producing Streptococcus pneumoniae cells in a pneumococcal growth media. Specifically, it has been discovered that the addition of potassium phosphate and calcium chloride to a pneumococcal growth media increases capsular polysaccharide titer.

[0056] In some embodiments, the medium described herein comprising potassium phosphate and calcium chloride is a pneumococcal growth medium. In some embodiments, the medium described herein comprising potassium phosphate and calcium chloride is a defined pneumococcal growth medium. In some embodiments, a known pneumococcal growth medium is modified to comprise potassium phosphate and calcium chloride at the concentrations provided herein.

[0057] In some embodiments, the components of the defined medium for culturing polysaccharide producing bacterial cells are provided as a powder formulation. In these embodiments, the powder ingredients will be added to a liquid cell culture medium preparation in order to obtain the recited concentration in moles or grams per liter of medium. In other embodiments, the components of the defined medium for culturing polysaccharide producing bacterial cells are provided in a liquid stock solution. In these embodiments, the liquid stock will be added to a liquid cell culture medium preparation in order to obtain the recited concentration in moles or grams per liter of medium.

[0058] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises potassium. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises a compound comprising potassium, (i.e., potassium phosphate, potassium carbonate, potassium bicarbonate, or potassium citrate). In particular embodiments, the medium for culturing polysaccharide producing bacterial cells comprises potassium phosphate (KH2PO4). In another particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises potassium phosphate monobasic.

[0059] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises potassium phosphate at a concentration between about 0.1 grams per liter of medium (g / L) and about 100 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises potassium phosphate at a concentration between about 0.1 g / L and about 10 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises potassium phosphate at a concentration of about 0.5 g / L, about 1 g / L, about 1 .5 g / L, about 2 g / L, about 2.5 g / L, about 3 g / L, about 3.5 g / L, about 4 g / L, about 4.5 g / L, or about 5 g / L.

[0060] In a particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises potassium phosphate at a concentration of about 1 g / L. In another particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises potassium phosphate at a total concentration of about 1 g / L and further comprises each of the following components: glucose, tyrosine, asparagine, sodium chloride, cysteine, glutamine, and iron sulfate. In another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises potassium phosphate at a total concentration of about 1 g / L and further comprises each of the following components: glucose, tyrosine, asparagine, sodium chloride, cysteine, glutamine, and iron sulfate.

[0061] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises calcium. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises a compound comprising calcium, (i.e., calcium chloride or calcium hydroxide). In particular embodiments, the medium for culturing polysaccharide producing bacterial cells comprises calcium chloride (CaCl2). In another particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises anhydrous calcium chloride.

[0062] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises calcium chloride at a concentration between about 0.01 g / L and about 10 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises calcium chloride at a concentration between about 0.1 g / L and about 1 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises calcium chloride at a concentration of about 0.05 g / L, about 0.1 g / L, about 0.15 g / L, about 0.2 g / L, about 0.25 g / L, about 0.3 g / L, about 0.35 g / L, about 0.4 g / L, about 0.45 g / L, or about 0.5 g / L. In other embodiments, the medium for culturing polysaccharide producing bacterial cells comprises calcium chloride at a concentration between about 0.1 g / L and about 0.2 g / L. In still other embodiments, the medium for culturing polysaccharide producing bacterial cells comprises calcium chloride at a concentration of about 0.1 g / L, about 0.11 g / L, about 0.12 g / L, about 0.13 g / L, about 0.14 g / L, about 0.15 g / L, about 0.16 g / L, about 0.17 g / L, about 0.18 g / L, about 0.19, or about 0.2 g / L.

[0063] In a particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L. In one particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises calcium chloride at a concentration of about 0.13 g / L. In another particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises calcium chloride at a concentration of about 0.13374 g / L.

[0064] In another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumonia cells comprises calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L, and potassium phosphate at a concentration of about 1 g / L. In still another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, and each of the following components: glucose, tyrosine, asparagine, sodium chloride, cysteine, glutamine, and iron sulfate.

[0065] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises tyrosine. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises L-tyrosine. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises L-Tyrosine disodium dihydrate (C9HgNNa2O3*2H2O).

[0066] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises Tyrosine disodium dihydrate at a concentration between about 0.01 grams per liter of medium (g / L) and about 10 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises Tyrosine disodium dihydrate at a concentration between about 0.1 g / L and about 1 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises Tyrosine disodium dihydrate at a concentration between about 0.6 g / L and about 0.7 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises Tyrosine disodium dihydrate at a concentration of about 0.6 g / L, about 0.61 g / L, about 0.62 g / L, about 0.63 g / L, about 0.64 g / L, about 0.65 g / L, about 0.66 g / L, about 0.67 g / L, about 0.68 g / L, about 0.69 g / L, or about 0.7 g / L.

[0067] In a particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises L-Tyrosine disodium dihydrate at a concentration of about 0.64 g / L. In one particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises L-Tyrosine disodium dihydrate at a concentration of about 0.643 g / L.

[0068] In another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumonia cells comprises calcium chloride at a concentration of between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, and L-Tyrosine disodium dihydrate at a concentration between about 0.6 g / L and about 0.7 g / L. In still another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration of between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, L-Tyrosine disodium dihydrate at a concentration between about 0.6 g / L and about 0.7 g / L, and each of the following components: glucose, asparagine, sodium chloride, cysteine, glutamine, and iron sulfate.

[0069] Any carbon source may be used in the culture medium of the present disclosure. Suitable carbon sources include glucose, dextrose, mannitol, lactose, sucrose, fructose, galactose, raffinose, xylose, and / or mannose. In a particular embodiment, the carbon source in the culture medium is glucose.

[0070] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises glucose. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises D-glucose (C6H12O6).

[0071] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises glucose at a concentration between about 1 g / L and about 100 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises glucose at a concentration between about 10 g / L and about 100 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises glucose at a concentration between about 50 g / L and about 60 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises glucose at a concentration of about 50 g / L, about 51 g / L, about 52 g / L, about 53 g / L, about 54 g / L, about 55 g / L, about 56 g / L, about 57 g / L, about 58 g / L, about 59 g / L, or about 60 g / L.

[0072] In a particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises D-glucose at a concentration of about 55 g / L.

[0073] In another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, and D-glucose at a concentration of about 55 g / L. In still another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, D-glucose at a concentration of about 55 g / L, and each of the following components: tyrosine, asparagine, sodium chloride, cysteine, glutamine, and iron sulfate.

[0074] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises asparagine. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises L-asparagine. In further embodiments, the medium for culturing polysaccharide producing bacterial cells comprises L-asparagine monohydrate (C4H10N2O4).

[0075] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises asparagine monohydrate at a concentration between about 0.5 g / L and about 50 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises asparagine monohydrate at a concentration between about 0.5 g / L and about 10 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises asparagine monohydrate at a concentration between about 1 g / L and about 5 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises asparagine monohydrate at a concentration between about 2 g / L and about 3 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises asparagine monohydrate at a concentration of about 1 g / L, about 1 .25 g / L, about 1 .5 g / L, about 1 .75 g / L, about 2 g / L, about 2.25 g / L, about 2.5 g / L, about 2.75 g / L, about 3 g / L, about 3.25 g / L, about 3.5 g / L, about 3.75 g / L, about 4 g / L, about 4.25 g / L, about 4.5 g / L, about 4.75 g / L, or about 5 g / L. In a particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises L-asparagine monohydrate at a concentration of about 2.25 g / L.

[0076] In another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, and L-asparagine monohydrate at a concentration between about 2.2 g / L and about 2.3 g / L. In still another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, L-asparagine monohydrate at a concentration between about 2.2 g / L and about 2.3 g / L, and each of the following components: glucose, tyrosine, sodium chloride, cysteine, glutamine, and iron sulfate.

[0077] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises sodium. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises a compound comprising sodium, (i.e., sodium chloride, sodium pyruvate, or disodium phosphate). In particular embodiments, the medium for culturing polysaccharide producing bacterial cells comprises sodium chloride (NaCI).

[0078] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises sodium chloride at a concentration between about 0.5 g / L and about 50 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises sodium chloride at a concentration between about 0.5 g / L and about 10 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises sodium chloride at a concentration between about 1 g / L and about 5 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises sodium chloride at a concentration between about 0.5 g / L and about 1 .5 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises sodium chloride at a concentration of about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 1.1 g / L, about 1.2 g / L, about 1 .3 g / L, about 1 .4 g / L, or about 1 .5 g / L. In a particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises sodium chloride at a concentration between about 1 g / L and about 1 .2 g / L.

[0079] In a particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises sodium chloride at a concentration of about 1.1 g / L.

[0080] In another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, and sodium chloride at a concentration of about 1.1 g / L. In still another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, sodium chloride at a concentration of about 1.1 g / L, and each of the following components: glucose, tyrosine, asparagine, cysteine, glutamine, and iron sulfate.

[0081] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises cysteine. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises L- cysteine. In further embodiments, the medium for culturing polysaccharide producing bacterial cells comprises L-cysteine HCI monohydrate (HSCH2CH(NH2)COOH*HCI*H2O).

[0082] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises cysteine HCI monohydrate at a concentration between about 0.01 g / L and about 100 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises cysteine HCI monohydrate at a concentration between about 0.1 g / L and about 10 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises cysteine HCI monohydrate at a concentration between about 0.1 g / L and about 1 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises cysteine HCI monohydrate at a concentration of about 0.1 g / L, about 0.2 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, or about 1 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises cysteine HCI monohydrate at a concentration between about 0.2 g / L and about 0.3 g / L. In a particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises L-cysteine HCI monohydrate at a concentration of about 0.25 g / L. In another particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises L-cysteine HCI monohydrate at a concentration of about 0.2504 g / L.

[0083] In another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, and cysteine HCI monohydrate at a concentration between about 0.2 g / L and about 0.3 g / L. In still another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, cysteine HCI monohydrate at a concentration between about 0.2 g / L and about 0.3 g / L, and each of the following components: glucose, tyrosine, asparagine, sodium chloride, glutamine, and iron sulfate.

[0084] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises glutamine (C5H10N2O3). In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises L-glutamine.

[0085] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises glutamine at a concentration between about 0.01 g / L and about 100 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises glutamine at a concentration between about 0.5 g / L and about 10 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises glutamine at a concentration between about 0.5 g / L and about 2.5 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises glutamine at a concentration between about 1 g / L and about 2 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises glutamine at a concentration of about 1 g / L, about 1.1 g / L, about 1.2 g / L, about 1.3 g / L, about 1.4 g / L, about 1 .5 g / L, about 1 .6 g / L, about 1 .7 g / L, about 1 .8 g / L, about 1 .9 g / L, or about 2 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises glutamine at a concentration between about 1.1 g / L and about 1 .2 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises glutamine at a concentration of about 1.1 g / L, about 1.11 g / L, about 1.12 g / L, about 1.13 g / L, about 1.14 g / L, about 1.15 g / L, about 1.16 g / L, about 1.17 g / L, about 1.18 g / L, about 1.19 g / L, or about 1.2 g / L.

[0086] In a particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises L-glutamine at a concentration of about 1.17 g / L.

[0087] In another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, and L-glutamine at a concentration between about 1.1 g / L and about 1 .2 g / L. In still another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, L-glutamine at a concentration between about 1.1 g / L and about 1 .2 g / L, and each of the following components: glucose, tyrosine, asparagine, sodium chloride, cysteine, and iron sulfate.

[0088] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises iron. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises a compound comprising iron, (i.e., iron sulfate or ferrous oxide). In particular embodiments, the medium for culturing polysaccharide producing bacterial cells comprises iron(ll) sulfate heptahydrate (FeSO4*7H2O).

[0089] In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises iron(ll) sulfate heptahydrate at a concentration between about 0.00001 g / L and about 0.01 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises iron(ll) sulfate heptahydrate at a concentration between about 0.001 g / L and about 0.01 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises iron(ll) sulfate heptahydrate at a concentration between about 0.004 g / L and about 0.005 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises iron(ll) sulfate heptahydrate at a concentration of about 0.004 g / L, about 0.0041 g / L, about 0.0042 g / L, about 0.0043 g / L, about 0.0044 g / L, about 0.0045 g / L, about 0.0046 g / L, about 0.0047 g / L, about 0.0048 g / L, about 0.0049 g / L, or about 0.005 g / L. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises iron(ll) sulfate heptahydrate at a concentration between about 0.0041 g / L and about 0.0042 g / L.

[0090] In a particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises iron(ll) sulfate heptahydrate at a concentration of about 0.004 g / L. In another particular embodiment, the medium for culturing polysaccharide producing bacterial cells comprises iron(ll) sulfate heptahydrate at a concentration of about 0.00417 g / L.

[0091] In another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, and iron(ll) sulfate heptahydrate at a concentration between about 0.0041 and about 0.0042. In still another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, iron(ll) sulfate heptahydrate at a concentration between about 0.0041 and about 0.0042, and each of the following components: glucose, tyrosine, asparagine, sodium chloride, cysteine, and glutamine.

[0092] In yet another particular embodiment, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, D-glucose at a concentration of about 55 g / L, L- Tyrosine disodium dihydrate at a concentration between about 0.6 g / L and about 0.7 g / L, L-asparagine monohydrate at a concentration between about 2.2 g / L and about 2.3 g / L, sodium chloride at a concentration of about 1.1 g / L, cysteine HCI monohydrate at a concentration between about 0.2 g / L and about 0.3 g / L, L- glutamine at a concentration between about 1.1 g / L and about 1.2 g / L, and iron(ll) sulfate heptahydrate at a concentration between about 0.0041 and about 0.0042.

[0093] In another aspect, the culture medium may include various other factors known in the art to enhance growth, such as amino acids, vitamins, nucleosides, and inorganic salts. In particular embodiments, the medium for culturing polysaccharide producing bacterial cells comprises potassium phosphate, calcium chloride and at least one of the additional medium components provided in Table 1 below. In other particular embodiments, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises potassium phosphate, calcium chloride, and at least one of the additional medium components provided in Table 1 below. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises potassium phosphate, calcium chloride, and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 of the additional medium components provided in Table 1 below. In some embodiments, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises potassium phosphate, calcium chloride, and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 of the additional medium components provided in Table 1 below.

[0094] In some embodiments, the medium for culturing polysaccharide producing bacterial cells consists of potassium phosphate, calcium chloride, and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 of the additional medium components provided in Table 1 below. In some embodiments, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells consists of potassium phosphate, calcium chloride, and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 of the additional medium components provided in Table 1 below.

[0095] In particular embodiments, the medium for culturing polysaccharide producing bacterial cells comprises calcium chloride at a concentration of between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, and at least one of the additional medium components provided in Table 1 below. In other particular embodiments, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration of between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, and at least one of the additional medium components provided in Table 1 below. In some embodiments, the medium for culturing polysaccharide producing bacterial cells comprises calcium chloride at a concentration of between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 of the additional medium components provided in Table 1 below. In some embodiments, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells comprises calcium chloride at a concentration of between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, and at least 1 , 2, 3,

[0096] 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27,

[0097] 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49,

[0098] 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 of the additional medium components provided in Table 1 below.

[0099] In some embodiments, the medium for culturing polysaccharide producing bacterial cells consists of calcium chloride at a concentration of between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 of the additional medium components provided in Table 1 below. In some embodiments, the medium for culturing polysaccharide producing Streptococcus pneumoniae cells consists of calcium chloride at a concentration of between about 0.13 g / L and about 0.14 g / L, potassium phosphate at a concentration of about 1 g / L, and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 of the additional medium components provided in Table 1 below.

[0100] Table 1 : Additional Medium Components

[0101] Furthermore, one of ordinary skill in the art will recognize that any of the conditions listed above may be used either singly or in various combinations with one another. By utilizing media formulation which exhibit one, some, or all of the above characteristics, one of ordinary skill in the art will be able to optimize cell growth and / or viability and to maximize the production of polysaccharide.

[0102] Any of these media formulations presented in the present disclosure may optionally be supplemented as necessary with particular ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, trace elements (inorganic compounds usually present at very low final concentrations), amino acids, lipids, protein hydrolysates, or glucose or other energy source. These optional supplements may be added at the beginning of the culture or may be added at a later point in order to replenish depleted nutrients or for another reason. One of ordinary skill in the art will be aware of any desirable or necessary supplements that may be included in the disclosed media formulations.

[0103] In another aspect, the media disclosed herein is for use in a method of increasing the capsular polysaccharide titer measured in grams per liter (g / L) produced by a bacterial cell culture over a unit of time. In some embodiments, the media disclosed herein is for use in a method of increasing the capsular polysaccharide titer measured in grams per liter (g / L) produced by a cell culture of Streptococcus pneumoniae over a unit of time. In some embodiments, the media disclosed herein is for use in a method of increasing the capsular polysaccharide titer measured in grams per liter (g / L) produced by a bacterial cell culture over a unit of time, wherein the unit of time is between about 1 hour and about 72 hours. In some embodiments, the unit of time is between about 15 hours and about 40 hours. For example, in some embodiments, the unit of time is about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, or about 32 hours. In particular embodiments, the unit of time is between about 20 hours and about 26 hours. In another particular embodiment, the unit of time is about 24 hours.

[0104] In some embodiments, the media disclosed herein is for use in a method of increasing the capsular polysaccharide titer measured in grams per liter (g / L) produced by a bacterial cell culture over a unit of time, wherein the polysaccharide titer is between about 0.5 g / L and about 5 g / L. For example, in some embodiments, the polysaccharide titer is at least about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 1.1 g / L, about 1.2 g / L, about 1.3 g / L, about 1 .4 g / L, about 1 .5 g / L, about 1 .6 g / L, about 1 .7 g / L, about 1 .8 g / L, about 1 .9 g / L, about 2 g / L, about 2.1 g / L, about 2.2 g / L, about 2.3 g / L, about 2.4 g / L, about 2.5 g / L, about 2.6 g / L, about 2.7 g / L, about 2.8 g / L, about 2.9 g / L, or about 3 g / L. In a particular embodiment, the polysaccharide titer is at least about 1 g / L. In another particular embodiment, the polysaccharide titer is at least about 2 g / L. In another particular embodiment, the cell culture comprises Streptococcus pneumoniae and the polysaccharide titer is at least about 1 g / L. In another particular embodiment, the cell culture comprises Streptococcus pneumoniae and the polysaccharide titer is at least about 2 g / L.

[0105] In another particular embodiment, the media disclosed herein is for use in a method of increasing the capsular polysaccharide titer measured in grams per liter (g / L) produced by a bacterial cell culture over a unit of time, wherein the polysaccharide titer is at least about 1 g / L and the unit of time is about 24 hours. In yet another particular embodiment, the media disclosed herein is for use in a method of increasing the capsular polysaccharide titer measured in grams per liter (g / L) produced by a bacterial cell culture over a unit of time, wherein the polysaccharide titer is at least about 2 g / L and the unit of time is about 24 hours. In a further particular embodiment, the media disclosed herein is for use in a method of increasing the capsular polysaccharide titer measured in grams per liter (g / L) produced by a cell culture of Streptococcus pneumoniae over a unit of time, wherein the polysaccharide titer is at least about 1 g / L and the unit of time is about 24 hours. In yet another particular embodiment, the media disclosed herein is for use in a method of increasing the capsular polysaccharide titer measured in grams per liter (g / L) produced by a cell culture of Streptococcus pneumoniae over a unit of time, wherein the polysaccharide titer is at least about 2 g / L and the unit of time is about 24 hours.

[0106] In another aspect, the media disclosed herein is for use in a method of increasing capsular polysaccharide specific productivity measured in grams polysaccharide per liter per OD (g / L / OD) of a bacterial cell culture over a unit of time. In a further aspect, the media disclosed herein is for use in a method of increasing capsular polysaccharide specific productivity measured in grams polysaccharide per liter per OD (g / L / OD) of a cell culture of Streptococcus pneumoniae over a unit of time. In some embodiments, the media disclosed herein is for use in a method of increasing capsular polysaccharide specific productivity measured in grams polysaccharide per liter per OD (g / L / OD) of a bacterial cell culture over a unit of time, wherein the unit of time is between about 1 hour and about 72 hours. In some embodiments, the unit of time is between about 15 hours and about 40 hours. For example, in some embodiments, the unit of time is about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, or about 32 hours. In particular embodiments, the unit of time is between about 20 hours and about 26 hours. In another particular embodiment, the unit of time is about 24 hours.

[0107] In some embodiments, the media disclosed herein is for use in a method of increasing capsular polysaccharide specific productivity measured in grams polysaccharide per liter per OD (g / L / OD) of a bacterial cell culture over a unit of time, wherein the polysaccharide specific productivity is between about 0.05 g / L / OD and about 0.5 g / L / OD. In other embodiments, the polysaccharide specific productivity is between about 0.1 g / L / OD and about 0.3 g / L / OD. For example, in some embodiments, the polysaccharide specific productivity is about 0.1 , about 0.11 , about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.2, about 0.21 , about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, or about 0.3 g / L / OD.

[0108] In particular embodiments, the media disclosed herein is for use in a method of increasing capsular polysaccharide specific productivity measured in grams polysaccharide per liter per OD (g / L / OD) of a bacterial cell culture over a unit of time, wherein the polysaccharide specific productivity is between about 0.1 g / L / OD and about 0.3 g / L / OD, and the unit of time is about 24 hours. In further particular embodiments, the media disclosed herein is for use in a method of increasing capsular polysaccharide specific productivity measured in grams polysaccharide per liter per OD (g / L / OD) of a cell culture of Streptococcus pneumoniae over a unit of time, wherein the polysaccharide specific productivity is between about 0.1 g / L / OD and about 0.3 g / L / OD, and the unit of time is about 24 hours.

[0109] Fermentation Methods

[0110] The present disclosure provides fermentation methods for cultivating polysaccharide producing bacteria. In one aspect, the cultivation methods of the present disclosure are used in combination with the media or backpressure methods described herein to maximize polysaccharide production. In one embodiment, growth of polysaccharide producing bacteria in the methods of the disclosure proceeds in at least two phases: seed growth and fermentation. A seed culture is first grown by inoculation from a stock culture, e.g., a working cell bank. The seed is used either to inoculate a second seed culture or to inoculate a relatively large fermentation culture. As is understood in the art, the number of seed cultures used may depend, for example, on the size and volume of the fermentation step.

[0111] Accordingly, in one aspect, the disclosure relates to a method of culturing polysaccharide producing bacteria. The method includes culturing a polysaccharide- producing bacterial cell in a first culture medium under conditions that facilitate growth of the cell; inoculating a second culture medium with all or a portion of said first medium after said first culturing; culturing said inoculated second medium under conditions that facilitate cell growth and / or polysaccharide production. The method may further include isolating a polysaccharide from said second medium. In one embodiment, the polysaccharide producing bacteria are grown in a first culture medium referred to as a seed culture. In one embodiment, the seed culture includes a culture medium as described above and an inoculation from a stock culture that was grown in the medium. In one embodiment, the first and second culture medium are the same. In another embodiment, the first and second culture medium are different.

[0112] The seed growth phase (or phases) is generally carried out to scale-up the quantity of the microorganism from a stored culture, so that it can be used as an inoculant for the fermentation phase. The volume and quantity of viable cells used to inoculate the fermentation culture can be controlled more accurately if taken from an actively growing culture (e.g., a seed culture), rather than if taken from a stored culture.

[0113] In addition, more than one (e.g., two or three) seed growth phases can be used to scale-up the quantity of polysaccharide producing bacteria for inoculation of the fermentation medium. In some embodiments, growth of polysaccharide producing bacteria in the fermentation phase proceeds directly from the stored culture by direct inoculation.

[0114] To start the fermentation phase, a portion or all of a seed culture containing the polysaccharide producing bacteria may be used to inoculate a fermentation culture medium. An appropriate concentration of seed culture to use to inoculate fermentation medium can be determined by those of skill in this art.

[0115] Fermentation may be used to produce the maximum cell growth and / or polysaccharide production in a large-scale environment. In one embodiment, the polysaccharide producing bacteria are grown as a fermentation culture. In one embodiment, the fermentation culture was inoculated from a seed culture that was grown in the first medium and the fermentation culture is carried out in a second medium. In one embodiment, the first medium and the second medium are different. In another embodiment, the first medium and the second medium are the same.

[0116] In one embodiment, the polysaccharide producing bacterial cell is cultured in a fed batch culture system using the defined media described above. In a fed batch system, the culture is initiated with an inoculation of cells, supplemented with at least one nutrient added during the culture, and terminated with a single harvest of cells. In one embodiment, the nutrient is added at a constant rate.

[0117] In one aspect, the carbon source is the nutrient added during the culture. The carbon source may be any carbon source described above for the defined media. In a particular embodiment, the carbon source is glucose.

[0118] In one embodiment, the polysaccharide producing bacterial cell is cultured in a perfusion culture system. An advantage of a perfusion system is that fresh medium may be added continuously. In addition, metabolic waste products may be removed during production while maintaining cell viability in the system. The perfusion culture system may include providing fresh medium to the cells while simultaneously removing spent medium that is substantially free of cells or includes a substantially lower cell concentration than that in the bioreactor. In a perfusion culture, cells can be retained by, for example, filtration, ultrasonic filtration, centrifugation, or sedimentation.

[0119] In one embodiment, the spent media is separated from the cells and removed, while retaining the cells in or returning the cells to the bioreactor. The separation step may be a normal flow filter and / or a tangential flow filter. In one embodiment, said filtration system comprises a hollow fiber filter. In another embodiment, said filtration system comprises a flat-sheet cassette. In another embodiment, the cells are separated from the spent medium by a centrifugation step. In another embodiment, the cells are separated from the spent medium by an ultrasonic separation step. In another embodiment, the cells are separated from the spent medium via a sedimentation system.

[0120] In yet another aspect, the cultivation is carried out using any of the methods disclosed herein until the cell density, as determined by optical density (OD) at 600 nm, of the bacterial cell culture is at least about 4.0. In some embodiments, the cell density as determined by optical density (OD) at 600 nm is at least about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, about 10.0, about 11.0, about 12.0, about 13.0, about 14.0, about 15.0, about 16.0, about 17.0, about 18.0, about 19.0, or about 20.0. In a particular embodiment, the cultivation is carried out until the cell density is at least about 10.0 as determined by optical density (OD) at 600 nm.

[0121] In a further aspect, the cultivation is carried out using any of the methods disclosed herein until the cell density, as determined by optical density (OD) at 600 nm, of the bacterial cell culture increased by less than about 2, about 1 .5, about 1 , or about 0.5 over the course of an hour. In some embodiments, the cultivation is carried out using any of the methods disclosed herein until the cell density, as determined by optical density (OD) at 600 nm, of the bacterial cell culture increased by less than about 1 , about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, or about 0.1 over the course of an hour. In a particular embodiment, the cultivation is carried out using any of the methods disclosed herein until the cell density, as determined by optical density (OD) at 600 nm, of the bacterial cell culture increased by less than about 0.5 over the course of an hour.

[0122] In one aspect, the cultivation is carried out using any of the methods disclosed herein until the polysaccharide titer of the bacterial cell culture is between about 0.5 g / L and about 5 g / L. In one embodiment, the cultivation is carried out using any of the methods disclosed herein until the polysaccharide titer of the bacterial cell culture is at least about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 1.1 g / L, about 1 .2 g / L, about 1 .3 g / L, about 1 .4 g / L, about 1 .5 g / L, about 1 .6 g / L, about 1 .7 g / L, about 1 .8 g / L, about 1 .9 g / L, about 2 g / L, about 2.1 g / L, about 2.2 g / L, about 2.3 g / L, about 2.4 g / L, about 2.5 g / L, about 2.6 g / L, about 2.7 g / L, about 2.8 g / L, about 2.9 g / L, or about 3 g / L. In a particular embodiment, the cultivation is carried out using any of the methods disclosed herein until the polysaccharide titer of the bacterial cell culture is at least about 1 g / L. In another particular embodiment, the cultivation is carried out using any of the methods disclosed herein until the polysaccharide titer of the bacterial cell culture is at least about 2 g / L. In a particular embodiment, the cultivation is carried out using any of the methods disclosed herein until the polysaccharide titer of the Streptococcus pneumoniae cell culture is at least about 1 g / L. In another particular embodiment, the cultivation is carried out using any of the methods disclosed herein until the polysaccharide titer of the Streptococcus pneumoniae cell culture is at least about 2 g / L.

[0123] In some embodiments, the bioreactor can contain a maximum of between about 500 mL and about 20,000 L of medium. In some embodiments, the bioreactor can contain a maximum of about 5 L, about 10 L, about 15 L, about 20 L, about 25 L, about 30 L, about 35 L, about 40 L, about 45 L, about 50 L, about 55 L, about 60 L, about 65 L, about 70 L, about 75 L, about 80 L, about 85 L, about 90 L, about 95 L, or about 100 L of medium. In a particular embodiment, the bioreactor can contain a maximum of about 10 L of medium. In another particular embodiment, the bioreactor can contain a maximum of about 75 L of medium.

[0124] In other embodiments, the bioreactor can contain a large volume of medium for manufacturing scale. In some embodiments, the bioreactor can contain a maximum of about 500 L, about 1000 L, about 1500 L, about 2000 L, about 2500 L, about 3000 L, about 3500 L, about 4000 L, about 4500 L, or about 5000 L of medium. In a particular embodiment, the bioreactor can contain a maximum of about 2000 L of medium.

[0125] Pressurized Fermentation Tank

[0126] The process of bacterial fermentation is facilitated when a high concentration of carbon dioxide is maintained within the fermentation medium. Traditionally, methods to increase the concentration of carbon dioxide within the fermentation medium included the use of a nitrogen overlay and the addition of increased concentrations of carbon containing components within the fermentation medium. Nitrogen overlay describes a method wherein nitrogen gas flows into a fermentation tank that is holding the fermenting bacteria. The nitrogen overlay method is typically performed within a fermentation system that is not airtight, such that the backpressure within the fermentation tank does not substantially increase during the fermentation process.

[0127] Disclosed herein are methods to ferment a bacterial cell culture in a pressurized bioreactor, i.e, fermentation tank. A bioreactor is said to be “pressurized” or contain “backpressure” when the pressure within the bioreactor is greater than atmospheric pressure, i.e., higher than 0 pound per square inch (PSI). Without being confined to a particular method or mechanism, it is hypothesized that fermentation is enhanced within a pressurized bioreactor due to the ability of the pressurized conditions to maintain a high level of carbon dioxide within the fermentation medium. For example, fermentation may be enhanced when the lag phase of the fermenting bacteria is decreased. “Lag phase” is a period of time when bacterial growth and division is arrested (see e.g., Bertrand et al. J Bacteriol. 2019 Mar 13;201 (7):e00697- 18). The longer the lag phase of the bacteria, the longer the total fermentation time for the bacterial culture to produce a target polysaccharide yield.

[0128] In some embodiments, culturing a bacterial cell culture under pressurized conditions decreases the lag phase of the bacteria during fermentation. In some embodiments, culturing a bacterial cell culture under pressurized conditions decreases the lag phase of the bacteria by between about 0.5 hours and about 15 hours. In some embodiments, culturing a bacterial cell culture under pressurized conditions decreases the lag phase of the bacteria by about 0.5 hours, about 1 hour, about 1 .5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about

[0129] 6.5 hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours, about 10 hours, about 10.5 hours, about 11 hours, about

[0130] 11 .5 hours, or about 12 hours. In further embodiments, culturing a bacterial cell culture under pressurized conditions decreases the lag phase of the bacteria by between about 1 and about 5 hours. In additional embodiments, culturing a bacterial cell culture under pressurized conditions decreases the lag phase of the bacteria by at least about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours. In a particular embodiment, culturing a Streptococcus pneumoniae cell culture under pressurized conditions decreases the lag phase of the bacteria by about 1 hour. In yet another particular embodiment, culturing a Streptococcus pneumoniae cell culture under pressurized conditions decreases the lag phase of the bacteria by about 2 hours. In still another particular embodiment, culturing a Streptococcus pneumoniae cell culture under pressurized conditions decreases the lag phase of the bacteria by about 3 hours. In another particular embodiment, culturing a Streptococcus pneumoniae cell culture under pressurized conditions decreases the lag phase of the bacteria by about 4 hours. In some embodiments, culturing a bacterial cell culture under pressurized conditions decreases the total culturing time required to achieve a target polysaccharide yield. In some embodiments, culturing a bacterial cell culture under pressurized conditions decreases the total culturing time required to achieve a target polysaccharide yield by between about 1 hour and about 20 hours. In some embodiments, culturing a bacterial cell culture under pressurized conditions decreases the total culturing time required to achieve a target polysaccharide yield by about 0.5 hours, about 1 hour, about 1 .5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours, about 10 hours, about 10.5 hours, about 11 hours, about 11 .5 hours, or about 12 hours. In a particular embodiment, culturing a Streptococcus pneumoniae cell culture under pressurized conditions decreases the total culturing time required to achieve a target polysaccharide yield by about 1 hour. In yet another particular embodiment, culturing a Streptococcus pneumoniae cell culture under pressurized conditions decreases the total culturing time required to achieve a target polysaccharide yield by about 2 hours. In still another particular embodiment, culturing a Streptococcus pneumoniae cell culture under pressurized conditions decreases the total culturing time required to achieve a target polysaccharide yield by about 3 hours. In another particular embodiment, culturing a Streptococcus pneumoniae cell culture under pressurized conditions decreases the total culturing time required to achieve a target polysaccharide yield by about 4 hours.

[0131] In some embodiments, the target polysaccharide yield is between about 1 and 10 grams of polysaccharide per liter. For example, in the some embodiments, the target polysaccharide yield is about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 grams per liter (g / L). In a particular embodiment, the target polysaccharide yield is between about 1 (g / L) and about 3 (g / L). In one particular embodiment, the target polysaccharide yield is about 1 (g / L). In another particular embodiment, the target polysaccharide yield is about 1 .5 (g / L). In yet another particular embodiment, the target polysaccharide yield is about 2 (g / L). In still another particular embodiment, the target polysaccharide yield is about 2.5 (g / L). In still other particular embodiments, culturing a bacterial cell culture under pressurized conditions decreases the total culturing time required to achieve a target polysaccharide yield by about 1 hour, about 2 hours, about 3 hours, or about 4 hours, wherein the target polysaccharide yield is at least about 1 (g / L). In another particular embodiment, culturing a bacterial cell culture under pressurized conditions decreases the total culturing time required to achieve a target polysaccharide yield by about 1 hour, about 2 hours, about 3 hours, or about 4 hours, wherein the target polysaccharide yield is at least about 2 (g / L).

[0132] In some embodiments, the method for producing bacterial capsular polysaccharides comprises the use of a pressurized bioreactor. In some embodiments, the method for producing bacterial capsular polysaccharides comprises the use of a pressurized bioreactor with a pressure greater than atmospheric pressure. In some embodiments, the method for producing bacterial capsular polysaccharides comprises the use of a pressurized bioreactor with a backpressure between about 0 PSI and about 1 PSI. In some embodiments, the method for producing bacterial capsular polysaccharides comprises the use of a pressurized bioreactor with a backpressure of at least about 1 PSI. In some embodiments, the method for producing bacterial capsular polysaccharides comprises the use of a pressurized bioreactor with a backpressure of at least about 5 PSI. In one embodiment, the method for producing bacterial capsular polysaccharides comprises the use of a pressurized bioreactor with a backpressure of at least about 10 PSI, about 15 PSI, or about 20 PSI. In exemplary embodiments, the method for producing bacterial capsular polysaccharides comprises the use of a pressurized bioreactor with a backpressure selected from the group consisting of about 1 PSI, about 2 PSI, about 3 PSI, about 4 PSI, about 5 PSI, about 6 PSI, about 7 PSI, about 8 PSI, about 9 PSI, or about 10 PSI. In a particular embodiment, the method for producing bacterial capsular polysaccharides comprises the use of a pressurized bioreactor with a backpressure of about 5 PSI. In another particular embodiment, the method for producing Streptococcus pneumoniae capsular polysaccharides comprises the use of a pressurized bioreactor with a backpressure of about 5 PSI.

[0133] In some embodiments, the method for producing bacterial capsular polysaccharides comprises the use of nitrogen overlay without a pressurized bioreactor. In additional embodiments, the method for producing bacterial capsular polysaccharides comprises the use of nitrogen overlay and a bioreactor with a backpressure of less than about 1 PSI.

[0134] In some embodiments, the method for producing bacterial capsular polysaccharides comprises the use of both nitrogen overlay and a pressurized bioreactor. In some embodiments, the method for producing bacterial capsular polysaccharides comprises the use of both nitrogen overlay and a pressurized bioreactor with a backpressure of at least about 1 PSI. In one embodiment, the method for producing bacterial capsular polysaccharides comprises the use of both nitrogen overlay and a pressurized bioreactor with a backpressure of at least about 10 PSI, about 15 PSI, or about 20 PSI. In exemplary embodiments, the method for producing bacterial capsular polysaccharides comprises the use of both nitrogen overlay and a pressurized bioreactor with a backpressure selected from the group consisting of about 1 PSI, about 2 PSI, about 3 PSI, about 4 PSI, about 5 PSI, about 6 PSI, about 7 PSI, about 8 PSI, about 9 PSI, or about 10 PSI. In a particular embodiment, the method for producing bacterial capsular polysaccharides comprises the use of both nitrogen overlay and a pressurized bioreactor with a backpressure of about 5 PSI.

[0135] In still another particular embodiment, culturing a bacterial cell culture under backpressure conditions of about 3 PSI, about 4 PSI or about 5 PSI decreases the lag phase of the bacteria by about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours. In yet another particular embodiment, culturing a bacterial cell culture under backpressure conditions of about 3 PSI, about 4 PSI or about 5 PSI decreases the total culturing time required to achieve a target polysaccharide yield by about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours. In another particular embodiment, culturing a bacterial cell culture under backpressure conditions of about 5 PSI decreases the lag phase of the bacteria by between about 1 and about 4 hours. In still another particular embodiment, culturing a bacterial cell culture under backpressure conditions of about 5 PSI decreases the total culturing time required to achieve a target polysaccharide yield by between about 1 and about 12 hours. In another particular embodiment, culturing a Streptococcus pneumoniae cell culture under backpressure conditions of about 5 PSI decreases the total culturing time required to achieve a target polysaccharide yield by between about 1 and about 12 hours.

[0136] In one aspect, a bacterial cell culture is cultured in a bioreactor with a backpressure of about 1 PSI, about 2 PSI, about 3 PSI, about 4 PSI, or about 5 PSI, and the bacterial cell culture is cultured in a medium comprising a compound comprising potassium and a compound comprising calcium. In a particular embodiment, a bacterial cell culture is cultured in a bioreactor with a backpressure of about 5 PSI, and the bacterial cell culture is cultured in a medium comprising a compound comprising potassium and a compound comprising calcium.

[0137] In one aspect, a Streptococcus pneumoniae cell culture is cultured in a bioreactor with a backpressure of about 1 PSI, about 2 PSI, about 3 PSI, about 4 PSI, or about 5 PSI, and the Streptococcus pneumoniae cell culture is cultured in a medium comprising potassium phosphate and calcium chloride. In a particular embodiment, a Streptococcus pneumoniae cell culture is cultured in a bioreactor with a backpressure of about 5 PSI, and the Streptococcus pneumoniae cell culture is cultured in a medium comprising potassium phosphate and calcium chloride. In some embodiments, the concentration of the potassium phosphate is between about 0.5 g / L and about 2 g / L. In a particular embodiment, the concentration of the potassium phosphate is about 1 g / L. In some embodiments, the concentration of the calcium chloride is between about 0.1 g / L and about 0.2 g / L. In a particular embodiment, the concentration of the calcium chloride is between about 0.13 g / L and about 0.14 g / L.

[0138] In one aspect, a Streptococcus pneumoniae cell culture is cultured in a bioreactor with a backpressure of about 1 PSI, about 2 PSI, about 3 PSI, about 4 PSI, or about 5 PSI, and the Streptococcus pneumoniae cell culture is cultured in a medium comprising potassium phosphate at a concentration of about 1 g / L and calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L. In a particular embodiment, a Streptococcus pneumoniae cell culture is cultured in a bioreactor with a backpressure of about 5 PSI, and the Streptococcus pneumoniae cell culture is cultured in a medium comprising potassium phosphate at a concentration of about 1 g / L and calcium chloride at a concentration between about 0.13 g / L and about 0.14 g / L.

[0139] In other embodiments, a bacterial cell culture of Streptococcus pneumoniae expressing serotype 15A is cultured in a bioreactor with a backpressure of about 1 PSI, about 2 PSI, about 3 PSI, about 4 PSI, or about 5 PSI. In a particular embodiment, a bacterial cell culture of Streptococcus pneumoniae expressing serotype 15A is cultured in a bioreactor with a backpressure of about 5 PSI.

[0140] In other embodiments, a bacterial cell culture of Streptococcus pneumoniae expressing serotype 23A is cultured in a bioreactor with a backpressure of about 1 PSI, about 2 PSI, about 3 PSI, about 4 PSI, or about 5 PSI. In a particular embodiment, a bacterial cell culture of Streptococcus pneumoniae expressing serotype 23A is cultured in a bioreactor with a backpressure of about 5 PSI.

[0141] In other embodiments, a bacterial cell culture of Streptococcus pneumoniae expressing serotype 23B is cultured in a bioreactor with a backpressure of about 1 PSI, about 2 PSI, about 3 PSI, about 4 PSI, or about 5 PSI. In a particular embodiment, a bacterial cell culture of Streptococcus pneumoniae expressing serotype 23B is cultured in a bioreactor with a backpressure of about 5 PSI.

[0142] In other embodiments, a bacterial cell culture of Streptococcus pneumoniae expressing serotype 24F is cultured in a bioreactor with a backpressure of about 1 PSI, about 2 PSI, about 3 PSI, about 4 PSI, or about 5 PSI. In a particular embodiment, a bacterial cell culture of Streptococcus pneumoniae expressing serotype 24F is cultured in a bioreactor with a backpressure of about 5 PSI.

[0143] In other embodiments, a bacterial cell culture of Streptococcus pneumoniae expressing serotype 35B is cultured in a bioreactor with a backpressure of about 1 PSI, about 2 PSI, about 3 PSI, about 4 PSI, or about 5 PSI. In a particular embodiment, a bacterial cell culture of Streptococcus pneumoniae expressing serotype 35B is cultured in a bioreactor with a backpressure of about 5 PSI.

[0144] Capsular Polysaccharides

[0145] Methods and materials for the production of bacterial capsular polysaccharides, in particular derived from S. pneumoniae are provided herein.

[0146] The term "saccharide" throughout this specification may indicate polysaccharide or oligosaccharide and includes both. In frequent embodiments, the saccharide is a polysaccharide, in particular a S. pneumoniae capsular polysaccharide.

[0147] S. pneumoniae capsular saccharides can be prepared by techniques known to those of ordinary skill in the art (see for example methods disclosed in US2006 / 0228380, US2006 / 0228381 , US2008 / 0102498, W02008 / 118752 and W02020170190). Typically, capsular polysaccharides are produced by growing each S. pneumoniae serotype in a medium (e.g., the medium disclosed herein), the polysaccharides are then prepared from the bacteria culture. Bacterial strains of S. pneumoniae used to make the respective capsular polysaccharides disclosed herein may be obtained from established culture collections (such as for example from the Streptococcal Reference Laboratory (Centers for Disease Control and Prevention, Atlanta, GA USA)) or clinical specimens.

[0148] The population of the organism (each S. pneumoniae serotype) increases from the initial seed vial to the eventual production scale fermenter. At the end of the growth cycle the cells are lysed and the lysate broth is then harvested for downstream (purification) processing (see for example WO 2006 / 110381 , WO 2008 / 118752, and U.S. Patent App. Pub. Nos. 2006 / 0228380, 2006 / 0228381 , 2008 / 0102498 and 2008 / 0286838).

[0149] The individual polysaccharides are typically purified through centrifugation, precipitation, ultra-filtration, and / or column chromatography (see for example WO 2006 / 110352, WO 2008 / 118752 and W02020170190).

[0150] An isolated capsular saccharide obtained by purification the S. pneumoniae lysate and optionally sized thereafter can be characterized by different parameters including, for example the weight average molecular weight (Mw). The molecular weight of the polysaccharide can be measured by Size Exclusion Chromatography (SEC) combined with Multiangle Laser Light Scattering detector (MALLS).

[0151] Purified polysaccharides may be activated (e.g., chemically activated) to make them capable of reacting (e.g., either directly to the carrier protein of via a linker such as an eTEC spacer) and then incorporated into glycoconjugates, as described herein.

[0152] In an embodiment, capsular saccharides of the disclosure may be one oligosaccharide unit, or a shorter than native length saccharide chain of repeating oligosaccharide units. In an embodiment, capsular saccharide of the disclosure is one repeating oligosaccharide unit of the relevant serotype.

[0153] In an embodiment, capsular saccharide of the disclosure may be oligosaccharides. Oligosaccharides have a low number of repeat units (typically 5-15 repeat units) and are typically derived synthetically or by hydrolysis of polysaccharides.

[0154] In an embodiment, the capsular saccharides of the present disclosure are polysaccharides. High molecular weight capsular polysaccharides are able to induce certain antibody immune responses due to the epitopes present on the antigenic surface. The isolation and purification of high molecular weight capsular polysaccharides is contemplated, for example, for use in the conjugates, compositions and methods of the present disclosure.

[0155] In order to generate capsular polysaccharides with advantageous filterability characteristics, immunogenicity and / or yields, sizing of the polysaccharide to a target molecular weight range can be performed prior to the conjugation to a carrier protein. Advantageously, the size of the purified capsular polysaccharide is reduced while preserving critical features of the structure of the polysaccharide. Mechanical or chemical sizing maybe employed.

[0156] In an embodiment, the size of the purified capsular polysaccharide is reduced by chemical hydrolysis. Chemical hydrolysis maybe conducted using a mild acid (e.g acetic acid, formic acid, propanoic acid). Chemical hydrolysis may also be conducted using a diluted strong acid (such as diluted hydrochloric acid, diluted sulfuric acid, diluted phosphoric acid, diluted nitric acid or diluted perchloric acid).

[0157] In particular embodiments, the size of the purified capsular polysaccharide is reduced by mechanical homogenization. In an embodiment, the size of the purified capsular polysaccharide is reduced by high pressure homogenization. High pressure homogenization achieves high shear rates by pumping the process stream through a flow path with sufficiently small dimensions. The shear rate is increased by using a larger applied homogenization pressure, and exposure time can be increased by recirculating the feed stream through the homogenizer.

[0158] Serotype 15A

[0159] In an aspect, the disclosure relates to capsular saccharides derived from S. pneumoniae serotype 15A. The structure of Streptococcus pneumoniae serotype 15A polysaccharide is known in the art (see e.g. Geno K et al. (2015) Clin Microbiol Rev Vol 28:3, p 871 -899).

[0160] In some embodiments, the isolated serotype 15A capsular polysaccharide (i.e. purified before further treatment) has a weight average molecular weight between 100 kDa and 2500 kDa. In an embodiment, the isolated serotype 15A capsular polysaccharide has a weight average molecular weight between 250 kDa and 1500 kDa. In a particular embodiment, the isolated serotype 15A capsular polysaccharide has a weight average molecular weight between 500 kDa and 1000 kDa. In an embodiment, the isolated serotype 15A capsular polysaccharide is sized to a weight average molecular weight of between 50 kDa and 500 kDa. In some embodiments, the isolated serotype 15A capsular polysaccharide is sized to a weight average molecular weight of between 75 kDa and 250 kDa. In particular embodiments, the isolated serotype 15A capsular polysaccharide is sized to a weight average molecular weight below 175 kDa. In another particular embodiment, the isolated serotype 15A capsular polysaccharide is sized to a weight average molecular weight of between 75 kDa and 175 kDa. In some embodiments, the isolated serotype 15A capsular polysaccharide is sized to a weight average molecular weight of between 100 kDa and 175 kDa.

[0161] In an embodiment, the isolated serotype 15A capsular polysaccharide is not sized.

[0162] The weight average molecular weight (Mw) of the saccharide before conjugation refers to the Mw before activation of the polysaccharide (i.e. after an eventual sizing step but before reacting the polysaccharide with an activating agent). In an embodiment, the isolated serotype 15A capsular polysaccharide before conjugation has a weight average molecular weight between 50 kDa and 500 kDa. In an embodiment, the isolated serotype 15A capsular polysaccharide before conjugation has a weight average molecular weight between 75 kDa and 250 kDa. In a particular embodiment, the isolated serotype 15A capsular polysaccharide before conjugation has a weight average molecular weight between 75 kDa and 175 kDa. In another particular embodiment, the isolated serotype 15A capsular polysaccharide before conjugation has a weight average molecular weight between 90 kDa and 150 kDa. In another particular embodiment, the isolated serotype 15A capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 175 kDa.

[0163] Serotype 23A

[0164] In an aspect the disclosure relates to capsular polysaccharides derived from S. pneumoniae serotype 23A. The structure of Streptococcus pneumoniae serotype 23A polysaccharide is known in the art (see e.g. Ravenscroft N et al. (2017) Carbohydrate Res. Vol. 450, p 19-29 and WO2019050814). The structure of the serotype 23A capsular polysaccharide is: —>4)-p-D-Glcp-(1—>3)-[[a-L-Rhap-(1—>2)]-[Gro-(2—>P—>3)]- P-D-Galp-(1 — >4)]-p-L-Rhap-(1 — In a particular embodiment, the isolated serotype 23A capsular polysaccharide (i.e. purified before further treatment) has a weight average molecular weight between 100 kDa and 2500 kDa. In an embodiment, the isolated serotype 23A capsular polysaccharide has a weight average molecular weight between 250 kDa and 1500 kDa. In an embodiment, the isolated serotype 23A capsular polysaccharide has a weight average molecular weight between 500 kDa and 1000 kDa.

[0165] In an embodiment, the isolated serotype 23A capsular polysaccharide is sized to a weight average molecular weight of between 50 kDa and 500 kDa. In a particular embodiment, the isolated serotype 23A capsular polysaccharide is sized to a weight average molecular weight of between 75 kDa and 400 kDa. In another particular embodiment, the isolated serotype 23A capsular polysaccharide is sized to a weight average molecular weight of between 100 kDa and 350 kDa. In another particular embodiment, the isolated serotype 23A capsular polysaccharide is sized to a weight average molecular weight of between 125 kDa and 225 kDa. For example, in some embodiments, the isolated serotype 23A polysaccharide is sized by mechanical homogenization, for example by high pressure homogenization.

[0166] In an embodiment, the isolated serotype 23A capsular polysaccharide is not sized.

[0167] In an embodiment, the isolated serotype 23A capsular polysaccharide before conjugation has a weight average molecular weight between 50 kDa and 500 kDa. In an embodiment, the isolated serotype 23A capsular polysaccharide before conjugation has a weight average molecular weight between 75 kDa and 400 kDa. In a particular embodiment, the isolated serotype 23A capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 350 kDa. In another particular embodiment, the isolated serotype 23A capsular polysaccharide before conjugation has a weight average molecular weight of between 125 kDa and 225 kDa.

[0168] Serotype 23B

[0169] In an aspect the disclosure relates to capsular saccharides derived from S. pneumoniae serotype 23B. The structure of Streptococcus pneumoniae serotype 23B polysaccharide is known in the art (see e.g. Ravenscroft N et al. (2017) Carbohydrate Res. Vol. 450, p 19-29 and WO2019050814). The structure of the serotype 23A capsular polysaccharide is: -^4)-[34D-Glcp-(1 — >4)-[Gro-(2— >P— >3)]- [3-D-Galp-(1 — >4)- P-L-Rhap-(1— >.

[0170] In a particular embodiment, the isolated serotype 23B capsular polysaccharide (i.e. purified before further treatment) has a weight average molecular weight between 100 kDa and 2500 kDa. In an embodiment, the isolated serotype 23B capsular polysaccharide has a weight average molecular weight between 250 kDa and 2000 kDa. In an embodiment, the isolated serotype 23B capsular polysaccharide has a weight average molecular weight between 300 kDa and 1000 kDa.

[0171] In an embodiment, the isolated serotype 23B capsular polysaccharide is sized to a weight average molecular weight of between 50 kDa and 750 kDa. In a particular embodiment, the isolated serotype 23B capsular polysaccharide is sized to a weight average molecular weight of between 75 kDa and 400 kDa. In another particular embodiment, the isolated serotype 23B capsular polysaccharide is sized to a weight average molecular weight of between 100 kDa and 250 kDa. In some embodiments, the isolated serotype 23B polysaccharide is sized by mechanical homogenization, for example by high pressure homogenization.

[0172] In an embodiment, the isolated serotype 23B capsular polysaccharide is not sized.

[0173] In an embodiment, the isolated serotype 23B capsular polysaccharide before conjugation has a weight average molecular weight between 50 kDa and 750 kDa. In an embodiment, the isolated serotype 23B capsular polysaccharide before conjugation has a weight average molecular weight between 75 kDa and 400 kDa. In a particular embodiment, the isolated serotype 23B capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 250 kDa.

[0174] Serotype 24F

[0175] In an aspect the disclosure relates to capsular saccharides derived from S. pneumoniae serotype 24F glycoconjugates. The structure of Streptococcus pneumoniae serotype 24F polysaccharide is known in the art (see e.g. WO201 9050815).

[0176] In a particular embodiment, the isolated serotype 24F capsular polysaccharide (i.e. purified before further treatment) has a weight average molecular weight between 100 kDa and 2500 kDa. In an embodiment, the isolated serotype 24F capsular polysaccharide has a weight average molecular weight between 250 kDa and 2000 kDa. In an embodiment, the isolated serotype 24F capsular polysaccharide has a weight average molecular weight between 500 kDa and 1000 kDa.

[0177] In an embodiment, the isolated serotype 24F capsular polysaccharide is sized to a weight average molecular weight of between 50 kDa and 500 kDa. In a particular embodiment, the isolated serotype 24F capsular polysaccharide is sized to a weight average molecular weight of between 75 kDa and 400 kDa. In another particular embodiment, the isolated serotype 24F capsular polysaccharide is sized to a weight average molecular weight of between 125 kDa and 275 kDa. In another particular embodiment, the isolated serotype 24F capsular polysaccharide is sized to a weight average molecular weight of between 125 kDa and 225 kDa. For example, the isolated serotype 24F polysaccharide is sized by mechanical homogenization, for example by high pressure homogenization.

[0178] In an embodiment, the isolated serotype 24F capsular polysaccharide is not sized.

[0179] In an embodiment, the isolated serotype 24F capsular polysaccharide before conjugation has a weight average molecular weight between 50 kDa and 500 kDa. In an embodiment, the isolated serotype 24F capsular polysaccharide before conjugation has a weight average molecular weight between 75 kDa and 400 kDa. In a particular embodiment, the isolated serotype 24F capsular polysaccharide before conjugation has a weight average molecular weight between 125 kDa and 275kDa. In another particular embodiment, the isolated serotype 24F capsular polysaccharide before conjugation has a weight average molecular weight between 125 kDa and 225 kDa.

[0180] Serotype 35B

[0181] In an aspect the disclosure relates to capsular saccharides derived from S. pneumoniae serotype 35B. The structure of Streptococcus pneumoniae serotype 35B polysaccharide is known in the art (see e.g. Geno K et al. (2015) Clin Microbiol Rev Vol 28:3, p 871 -899).

[0182] In a particular embodiment, the isolated serotype 35B capsular polysaccharide (i.e. purified before further treatment) has a weight average molecular weight between 100 kDa and 5000 kDa. In an embodiment, the isolated serotype 35B capsular polysaccharide has a weight average molecular weight between 300 kDa and 2000 kDa. In a particular embodiment, the isolated serotype 35B capsular polysaccharide has a weight average molecular weight between 500 kDa and 1000 kDa. In an embodiment, the isolated serotype 35B capsular polysaccharide is not sized.

[0183] In an embodiment, the isolated serotype 35B capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 5,000 kDa. In an embodiment, the isolated serotype 35B capsular polysaccharide before conjugation has a weight average molecular weight between 300 kDa and 2000 kDa. In a particular embodiment, the isolated serotype 35B capsular polysaccharide before conjugation has a weight average molecular weight between 500 kDa and 1000 kDa.

[0184] Serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 15B, 14, 18C, 19A, 19F, 22F, 23F and 33F

[0185] In an aspect the disclosure relates to capsular saccharides derived from S. pneumoniae serotype 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F.

[0186] The structures of Streptococcus pneumoniae serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F polysaccharides are known in the art (see e.g. Geno K et al. (2015) Clin Microbiol Rev Vol 28:3, p 871- 899).

[0187] In a particular embodiment, the isolated capsular polysaccharides (i.e. purified before further treatment) have a weight average molecular weight between 5 kDa and 5,000 kDa. In an embodiment, the isolated capsular polysaccharide has a weight average molecular weight between 10 kDa and 3,000 kDa. In an embodiment, the isolated capsular polysaccharide has a weight average molecular weight between 50 kDa and 1 ,000 kDa.

[0188] In an embodiment, the isolated serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F capsular polysaccharide is not sized.

[0189] In an embodiment, the isolated serotype 1 capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1000 kDa. In an embodiment, the isolated serotype 1 capsular polysaccharide before conjugation has a weight average molecular weight between 150 kDa and 900 kDa. In a particular embodiment, the isolated serotype 1 capsular polysaccharide before conjugation has a weight average molecular weight between 150 kDa and 700 kDa. In an embodiment, the serotype 3 capsular polysaccharide of the present disclosure comprises a serotype 3 capsular polysaccharide wherein the weight average molecular weight (Mw) of said polysaccharide before conjugation is between 50 kDa and 1 ,000 kDa. In a particular embodiment, the weight average molecular weight (Mw) is between 100 kDa and 300 kDa.

[0190] In an embodiment, the isolated serotype 4 capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1000 kDa. In an embodiment, the isolated serotype 4 capsular polysaccharide before conjugation has a weight average molecular weight between 300 kDa and 900 kDa.

[0191] In an embodiment, the isolated serotype 5 capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1 ,000 kDa. In an embodiment, the isolated serotype 5 capsular polysaccharide before conjugation has a weight average molecular weight between 200 kDa and 600 kDa.

[0192] In an embodiment, the isolated serotype 6A capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1000 kDa. In an embodiment, the isolated serotype 6A capsular polysaccharide before conjugation has a weight average molecular weight between 300 kDa and 900 kDa.

[0193] In an embodiment, the isolated serotype 6B capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1000 kDa. In an embodiment, the isolated serotype 6B capsular polysaccharide before conjugation has a weight average molecular weight between 200 kDa and 900 kDa.

[0194] In an embodiment, the isolated serotype 7F capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1000 kDa. In an embodiment, the isolated serotype 7F capsular polysaccharide before conjugation has a weight average molecular weight between 200 kDa and 900 kDa.

[0195] In an embodiment, the isolated serotype 8 capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1000 kDa. In an embodiment, the isolated serotype 8 capsular polysaccharide before conjugation has a weight average molecular weight between 200 kDa and 400 kDa.

[0196] In an embodiment, the isolated serotype 9V capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1000 kDa. In an embodiment, the isolated serotype 9V capsular polysaccharide before conjugation has a weight average molecular weight between 200 kDa and 900 kDa. In an embodiment, the isolated serotype 10A capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1000 kDa. In an embodiment, the isolated serotype 10A capsular polysaccharide before conjugation has a weight average molecular weight between 200 kDa and 900 kDa.

[0197] In an embodiment, the isolated serotype 11A capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1000 kDa. In an embodiment, the isolated serotype 11A capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 400 kDa.

[0198] In an embodiment, the isolated serotype 12F capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1000 kDa. In an embodiment, the isolated serotype 12F capsular polysaccharide before conjugation has a weight average molecular weight between 150 kDa and 400 kDa.

[0199] In an embodiment, the isolated serotype 14 capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1000 kDa. In an embodiment, the isolated serotype 14 capsular polysaccharide before conjugation has a weight average molecular weight between 200 kDa and 900 kDa.

[0200] In an embodiment, the isolated serotype 15B capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1000 kDa. In an embodiment, the isolated serotype 15B capsular polysaccharide before conjugation has a weight average molecular weight between 150 kDa and 300 kDa.

[0201] In an embodiment, the isolated serotype 18C capsular polysaccharide before conjugation has a weight average molecular weight between 20 kDa and 1000 kDa. In an embodiment, the isolated serotype 18C capsular polysaccharide before conjugation has a weight average molecular weight between 20 kDa and 500 kDa.

[0202] In an embodiment, the isolated serotype 19A capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1000 kDa. In an embodiment, the isolated serotype 19A capsular polysaccharide before conjugation has a weight average molecular weight between 250 kDa and 700 kDa.

[0203] In an embodiment, the isolated serotype 19F capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1000 kDa. In an embodiment, the isolated serotype 19F capsular polysaccharide before conjugation has a weight average molecular weight between 250 kDa and 800 kDa.

[0204] In an embodiment, the isolated serotype 22F capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1000 kDa. In an embodiment, the isolated serotype 22F capsular polysaccharide before conjugation has a weight average molecular weight between 400 kDa and 700 kDa.

[0205] In an embodiment, the isolated serotype 23F capsular polysaccharide before conjugation has a weight average molecular weight between 100 kDa and 1000 kDa. In an embodiment, the isolated serotype 23F capsular polysaccharide before conjugation has a weight average molecular weight between 200 kDa and 800 kDa.

[0206] In an embodiment, the isolated serotype 33F capsular polysaccharide before conjugation has a weight average molecular weight between 300 kDa and 2000 kDa. In an embodiment, the isolated serotype 33F capsular polysaccharide before conjugation has a weight average molecular weight between 500 kDa and 2000 kDa.

[0207] Glycoconjugates

[0208] In some embodiments, the capsular polysaccharides (CPS) produced using the medium and methods disclosed herein are conjugated (known as glycoconjugates). For the purpose of the disclosure, the term ‘glycoconjugate' indicates a capsular saccharide conjugated to a carrier protein via covalent or non-covalent bonds. In an embodiment, the capsular saccharide is conjugated to a carrier protein via non- covalent bonds (such as the rhizavidin / biotin system, see e.g. WO2012155007, W02020056202). In particular embodiments, the capsular saccharide is conjugated via covalent bonds. In one embodiment the capsular saccharide is conjugated directly to a carrier protein. In a second embodiment the capsular saccharide is conjugated to a carrier protein through a spacer / linker. A component of the glycoconjugate(s) of the disclosure is a carrier protein to which the saccharide is conjugated. The terms "protein carrier" or "carrier protein" or “carrier” may be used interchangeably herein. Carrier proteins should be amenable to standard conjugation procedures.

[0209] In an embodiment, the carrier protein of the glycoconjugate of the disclosure is: DT (Diphtheria Toxoid), TT (Tetanus Toxoid) or fragment C of TT, CRM197 (a nontoxic but antigenically identical variant of diphtheria toxin), other DT mutants (such as CRM176, CRM228, CRM45 (Uchida et al. (1973) J. Biol. Chem. 218:3838-3844), CRM9, CRM102, CRM103 or CRM107; and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc. (1992); deletion or mutation of Glu-148 to Asp, Gin or Ser and / or Ala 158 to Gly and other mutations disclosed in U.S. Patent Nos. 4,709,017 and 4,950,740; mutation 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 fragment disclosed in U.S. Patent No. 5,843,711 , pneumococcal pneumolysin (ply) (Kuo et al. (1995) Infect Immun 63:2706-2713) including ply detoxified in some fashion, for example 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 fusions of Pht proteins, for example PhtDE fusions, PhtBE fusions, Pht A-E (WO 01 / 98334, WO 03 / 054007, WO 2009 / 000826), OMPC (meningococcal outer membrane protein), which is usually extracted from Neisseria meningitidis serogroup B (EP0372501 ), PorB (from N. meningitidis), PD (Haemophilus influenzae protein D; see, e.g., EP0594610 B), or immunologically functional equivalents thereof, 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 comprising multiple human CD4+ T cell epitopes 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 proteins (WO 01 / 72337), toxin A or B of Clostridium difficile (WO 00 / 61761 ), transferrin binding proteins, pneumococcal adhesion protein (PsaA) or recombinant Pseudomonas aeruginosa exotoxin A (in particular non-toxic mutants thereof (such as exotoxin A bearing a substution at glutamic acid 553 (Douglas et al. (1987) J. Bacteriol. 169(11 ):4967-4971 )). Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD) also can be used as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins such as cholera toxoid (e.g., as described in WO 2004 / 083251 ), Escherichia coli LT, E. coli ST, and exotoxin A from P. aeruginosa. Another suitable carrier protein is a C5a peptidase from Streptococcus (SCP).

[0210] In an embodiment, the carrier protein of the glycoconjugates of the disclosure is the fusion protein CP1. The CP1 fusion protein comprises a biotin-binding protein such as, e.g ., a truncated rhizavidin protein (e.g, amino acids 45-179 of a wild-type rhizavidin protein), a first linker (e.g, a GGGGSSS linker), a SP1500 polypeptide (e.g., amino acids 27-278 of a full-length S. pneumoniae SP1500 polypeptide), a second linker (e.g, the amino acid sequence AAA), and a SP0785 polypeptide (e.g, amino acids 33-399 of a full length S. pneumoniae SP0785 polypeptide) see e.g. W02020056202.

[0211] In another embodiment, the carrier protein of the glycoconjugate of the disclosure is PD ( / - / . influenzae protein D; see, e.g., EP0594610 B).

[0212] In particular embodiments, the carrier protein of the glycoconjugates of the disclosure is DT, TT, CRM197 or a C5a peptidase from Streptococcus (SCP).

[0213] In an embodiment, the carrier protein of the glycoconjugate of the disclosure is DT (Diphtheria toxoid).

[0214] In another embodiment, the carrier protein of the glycoconjugate of the disclosure is TT (tetanus toxoid).

[0215] In a particular embodiment, the carrier protein of the glycoconjugate of the disclosure is CRMi9? or a C5a peptidase from Streptococcus (SCP).

[0216] In a particular embodiment, the carrier protein of the glycoconjugate of the disclosure is CRM197. The CRM197 protein is a nontoxic form of diphtheria toxin but is immunologically indistinguishable from the diphtheria toxin. CRM197 is produced by Corynebacterium diphtheriae infected by the nontoxigenic phage (3197tox-created by nitrosoguanidine mutagenesis of the toxigenic corynephage beta (llchida et al. (1971 ) Nature New Biology 233:8-11 ). The CRM197 protein has the same molecular weight as the diphtheria toxin but differs therefrom by a single base change (guanine to adenine) in the structural gene. This single base change causes an amino acid substitution (glutamic acid for glycine) in the mature protein and eliminates the toxic properties of diphtheria toxin. The CRM197 protein is a safe and effective T-cell dependent carrier for saccharides. Further details about CRM197 and production thereof can be found, e.g., in U.S. Patent No. 5,614,382.

[0217] In an embodiment, the carrier protein of the glycoconjugate of the disclosure is the A chain of CRM197 (see CN103495161 ). In an embodiment, the carrier protein of the glycoconjugate of the disclosure is the A chain of CRM197 obtained via expression by genetically recombinant E. coli (see CN103495161 ).

[0218] In other particular embodiments, the carrier protein of the glycoconjugate of the disclosure is SCP (Streptococcal C5a Peptidase). Two important species of [3- hemolytic streptococci, Streptococcus pyogenes (group A Streptococcus, GAS) and Streptococcus agalactiae (group B Streptococcus, GBS), which cause a variety of serious human infections that range from mild cases of pharyngitis and impetigo to serious invasive diseases such as necrotizing fasciitis (GAS) and neonatal sepsis (GBS) have developed a way to defeat this immune response. All human isolates of [3-hemolytic streptococci, including GAS and GBS, produce a highly conserved cellwall protein SCP (Streptococcal C5a Peptidase) that specifically inactivates C5a. The scp genes from GAS and GBS encode a polypeptide containing between 1 ,134 and 1 ,181 amino acids (Brown et al., PNAS, 2005, vol. 102, no. 51 pages 18391-18396). The first 31 residues are the export signal presequence and are removed upon passing through the cytoplasmic membrane. The next 68 residues serve as a prosequence and must be removed to produce active SCP. The next 10 residues can be removed without loss of protease activity. At the other end, starting with Lys-1034, are four consecutive 17-residue motifs followed by a cell sorting and cell-wall attachment signal. This combined signal is composed of a 20-residue hydrophilic sequence containing an LPTTND sequence, a 17-residue hydrophobic sequence, and a short basic carboxyl terminus.

[0219] SCP can be divided in domains (see figure 1 B of Brown etal., PNAS, 2005, vol. 102, no. 51 pages 18391-18396). These domains are the Pre / Pro domain (which comprises the export signal presequence (commonly the first 31 residues) and the pro-sequence (commonly the next 68 residues)), the protease domain (which is splitted in two part (protease part 1 commonly residues 89-333 / 334 and protease domain part 2 and commonly residues 467 / 468-583 / 584), the protease-associated domain (PA domain) (commonly residues 333 / 334-467 / 468), three fibronectin type III (Fn) domains (Fn1 , commonly residues 583 / 584-712 / 713; Fn2, commonly residues 712 / 713-928 / 929 / 930; commonly Fn3, residues 929 / 930-1029 / 1030 / 1031 ) and a cell wall anchor domain (commonly redisues 1029 / 1030 / 1031 to the C-terminus).

[0220] In an embodiment, the carrier protein of the glycoconjugate of the disclosure is an SCP from GBS (SCPB). An example of SCPB is provided at SEQ ID. NO: 3 of W097 / 26008. See also SEQ ID NO: 3 of WOOO / 34487.

[0221] In another particular embodiment, the carrier protein of the glycoconjugate of the disclosure is an SCP from GAS (SCPA). Examples of SCPA can be found at SEQ ID.No.1 and SEQ ID.No.2 of WQ97 / 26008. See also SEQ ID NO: 1 , 2 and 23 of WOOO / 34487.

[0222] In a particular embodiment, the carrier protein of the glycoconjugate of the disclosure is an enzymatically inactive SCP.

[0223] In other particular embodiments, the carrier protein of the glycoconjugate of the disclosure is an enzymatically inactive SCP from GBS (SCPB). In another particular embodiment, the carrier protein of the glycoconjugate of the disclosure is an enzymatically inactive SCP from GAS (SCPA).

[0224] In an embodiment, the carrier protein of the glycoconjugate of the disclosure is a fragment of an SCP. In an embodiment, the carrier protein of the glycoconjugate of the disclosure is a fragment of an SCPA. In another embodiment, the carrier protein of the glycoconjugate of the disclosure is a fragment of an SCPB.

[0225] If the protein carrier is the same for 2 or more saccharides in the composition, the saccharides can be conjugated to the same molecule of the protein carrier (carrier molecules having 2 or more different saccharides conjugated to it) [see for instance W02020121 159],

[0226] In a particular embodiment though, the saccharides are each individually conjugated to different molecules of the protein carrier (each molecule of protein carrier only having one type of saccharide conjugated to it). In said embodiment, the capsular saccharides are said to be individually conjugated to the carrier protein.

[0227] In a particular embodiment, a glycoconjugate is provided wherein a capsular polysaccharide derived from a S. pneumoniae serotype selected from the group consisting of 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and 35B is conjugated to CRM197.

[0228] In another particular embodiment, a glycoconjugate is provided wherein a capsular polysaccharide derived from a S. pneumoniae serotype selected from the group consisting of 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and 35B is conjugated to SCP.

[0229] In an embodiment, provided herein is an immunogenic composition comprising glycoconjugates from S. pneumoniae serotypes 15A, 23A, 23B, 24F, and 35B conjugated to CRM197.

[0230] In an embodiment, provided herein is an immunogenic composition comprising glycoconjugates from S. pneumoniae serotypes 15A, 23A, 23B, 24F, and 35B conjugated to SCP.

[0231] In an embodiment, provided herein is an immunogenic composition comprising glycoconjugates from S. pneumoniae serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and 35B conjugated to CRM197.

[0232] In an embodiment, provided herein is an immunogenic composition comprising glycoconjugates from S. pneumoniae serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and 35B conjugated to SCP.

[0233] In an embodiment, provided herein is an immunogenic composition comprising glycoconjugates from S. pneumoniae serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and 35B, wherein the glycoconjugate from S. pneumoniae serotype 3 is conjugated to SCP and the glycoconjugates from S. pneumoniae serotypes 1 , 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and 35B are conjugated to CRM197.

[0234] Immunogenic Compositions

[0235] The capsular polysaccharides (CPS) and / or glycoconjugates disclosed herein may be used as antigens in an immunogenic composition. For example, they may be part of a vaccine.

[0236] In various embodiments, the number of different S. pneumoniae capsular saccharides can range from 1 different serotype (or "v", valence) to 25 different serotypes (25v), or more.

[0237] In an embodiment the disclosure relates to an immunogenic composition comprising 1 to 25 different serotypes of S. pneumoniae of the disclosure (1 to 25 pneumococcal conjugates). For example, the immunogenic composition may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 different serotypes of S. pneumoniae of the disclosure. In a particular embodiment, the immunogenic composition comprises 25 different serotypes of S. pneumoniae of the disclosure.

[0238] In another particular embodiment, the immunogenic composition comprises S. pneumoniae serotypes 15A, 23A, 23B, 24F, and 35B.

[0239] In still another particular embodiment, the immunogenic composition comprises S. pneumoniae serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and 35B.

[0240] The "immunogenic amount" of the different saccharide components in the immunogenic composition, may diverge and each may comprise about 0.5 pg, about 0.75 pg, about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 15 pg, about 20 pg, about 30 pg, about 40 g, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, or about 100 pg of any particular saccharide antigen.

[0241] Generally, each dose will comprise 0.1 pg to 100 pg of saccharide for a given serotype. In a particular embodiment, each dose will comprise 0.5 pg to 20 pg of saccharide for a given serotype. In another particular embodiment, each dose will comprise 2.0 pg to 10.0 pg of saccharide for a given serotype.

[0242] In an embodiment, each dose will comprise about 0.5 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 1 .0 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 1.1 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 1.5 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 2.0 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 2.2 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 2.5 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 3.0 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 3.5 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 4.0 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 4.4 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 5.0 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 5.5 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 6.0 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 6.5 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 7.0 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 7.5 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 8.0 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 8.5 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 9.0 pg of saccharide for each particular glycoconjugate. In an embodiment, each dose will comprise about 0.5 pg, about 1.0 pg, about 1.5 pg, about 2.0 pg, about 2.2 pg, about 2.5 pg, about 3.0 pg, about 3.5 pg, about 4.0 pg, about 4.5 pg, or about 5.0 pg of polysaccharide for glycoconjugates from S. pneumoniae serotype 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and / or 35B.

[0243] The immunogenic compositions of the disclosure may be formulated in liquid form (i.e., solutions or suspensions) or in a lyophilized form. In an embodiment, the immunogenic composition of the disclosure is formulated in a liquid form. In an embodiment, the immunogenic composition of the disclosure is formulated in a lyophilized form. Liquid formulations may advantageously be administered directly from their packaged form and are thus ideal for injection without the need for reconstitution in aqueous medium as otherwise required for lyophilized compositions of the disclosure.

[0244] Formulation of the immunogenic composition of the present disclosure can be accomplished using art-recognized methods. For instance, the individual polysaccharides and / or conjugates can be formulated with a physiologically acceptable vehicle to prepare the composition. Examples of such vehicles include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.

[0245] The present disclosure provides an immunogenic composition comprising any combination of glycoconjugates disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0246] In an embodiment, the immunogenic composition of the disclosure is in liquid form, for example in aqueous liquid form.

[0247] Immunogenic compositions of the disclosure may comprise one or more of a buffer, a salt, a divalent cation, a non-ionic detergent, a cryoprotectant such as a sugar, and an anti-oxidant such as a free radical scavenger or chelating agent, or any multiple combinations thereof.

[0248] In an embodiment, the immunogenic compositions of the disclosure comprise a buffer. In an embodiment, said buffer has a pKa of about 3.5 to about 7.5. In some embodiments, the buffer is phosphate, succinate, histidine or citrate. In some embodiments, the buffer is succinate. In some embodiments, the buffer is histidine. In certain embodiments, the buffer is succinate at a final concentration of 1 mM to 10 mM. In one particular embodiment, the final concentration of the succinate buffer is about 5 mM.

[0249] In an embodiment, the immunogenic compositions of the disclosure comprise a salt. In some embodiments, the salt is selected from the groups consisting of magnesium chloride, potassium chloride, sodium chloride, and a combination thereof. In one particular embodiment, the salt is sodium chloride. In one particular embodiment, the immunogenic compositions of the disclosure comprise sodium chloride at 150 mM.

[0250] In an embodiment, the immunogenic compositions of the disclosure comprise a surfactant. In an embodiment, the surfactant is selected from the group consisting of polysorbate 20 (TWEENTM20), polysorbate 40 (TWEEN™40), polysorbate 60 (TWEENTM60), polysorbate 65 (TWEENTM65), polysorbate 80 (TWEENTM80), polysorbate 85 (TWEENTM85), TRITON™ N-101 , TRITON™ X-100, oxtoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate (PEG-15, Solutol H 15), polyoxyethylene-35-ricinoleate (CREMOPHOR® EL), soy lecithin, and a poloxamer.

[0251] In one particular embodiment, the surfactant is polysorbate 80. In some embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.0001 % to 10% polysorbate 80 weight to weight (w / w). In some embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.001 % to 1 % polysorbate 80 weight to weight (w / w). In some embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.01 % to 1 % polysorbate 80 weight to weight (w / w). In other embodiments, the final concentration of polysorbate 80 in the formulation is 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1 % polysorbate 80 (w / w). In another embodiment, the final concentration of the polysorbate 80 in the formulation is 0.02% polysorbate 80 (w / w). In another embodiment, the final concentration of the polysorbate 80 in the formulation is 0.01% polysorbate 80 (w / w). In another embodiment, the final concentration of the polysorbate 80 in the formulation is 0.03% polysorbate 80 (w / w). In another embodiment, the final concentration of the polysorbate 80 in the formulation is 0.04% polysorbate 80 (w / w). In another embodiment, the final concentration of the polysorbate 80 in the formulation is 0.05% polysorbate 80 (w / w). In another embodiment, the final concentration of the polysorbate 80 in the formulation is 1% polysorbate 80 (w / w). In one particular embodiment, the surfactant is polysorbate 20. In some embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.0001 % to 10% polysorbate 20 weight to weight (w / w). In some embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.001 % to 1 % polysorbate 20 weight to weight (w / w). In some embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.01 % to 1 % polysorbate 20 weight to weight (w / w). In other embodiments, the final concentration of polysorbate 20 in the formulation is 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1 % polysorbate 20 (w / w). In another embodiment, the final concentration of the polysorbate 20 in the formulation is 0.02% polysorbate 20 (w / w). In another embodiment, the final concentration of the polysorbate 20 in the formulation is 0.01% polysorbate 20 (w / w). In another embodiment, the final concentration of the polysorbate 20 in the formulation is 0.03% polysorbate 20 (w / w). In another embodiment, the final concentration of the polysorbate 20 in the formulation is 0.04% polysorbate 80 (w / w). In another embodiment, the final concentration of the polysorbate 20 in the formulation is 0.05% polysorbate 20 (w / w). In another embodiment, the final concentration of the polysorbate 20 in the formulation is 1% polysorbate 20 (w / w).

[0252] In one particular embodiment, the surfactant is polysorbate 40. In some embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.0001 % to 10% polysorbate 40 weight to weight (w / w). In some embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.001 % to 1 % polysorbate 40 weight to weight (w / w). In some embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.01 % to 1 % polysorbate 40 weight to weight (w / w). In other embodiments, the final concentration of polysorbate 40 in the formulation is 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1 % polysorbate 40 (w / w). In another embodiment, the final concentration of the polysorbate 40 in the formulation is 1 % polysorbate 40 (w / w).

[0253] In one particular embodiment, the surfactant is polysorbate 60. In some embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.0001 % to 10% polysorbate 60 weight to weight (w / w). In some embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.001 % to 1 % polysorbate 60 weight to weight (w / w). In some embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.01 % to 1 % polysorbate 60 weight to weight (w / w). In other embodiments, the final concentration of polysorbate 60 in the formulation is 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1 % polysorbate 60 (w / w). In another embodiment, the final concentration of the polysorbate 60 in the formulation is 1 % polysorbate 60 (w / w).

[0254] In one particular embodiment, the surfactant is polysorbate 65. In some embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.0001 % to 10% polysorbate 65 weight to weight (w / w). In some embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.001 % to 1 % polysorbate 65 weight to weight (w / w). In some embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.01 % to 1 % polysorbate 65 weight to weight (w / w). In other embodiments, the final concentration of polysorbate 65 in the formulation is 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1 % polysorbate 65 (w / w). In another embodiment, the final concentration of the polysorbate 65 in the formulation is 1 % polysorbate 65 (w / w).

[0255] In one particular embodiment, the surfactant is polysorbate 85. In some embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.0001 % to 10% polysorbate 85 weight to weight (w / w). In some embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.001 % to 1 % polysorbate 85 weight to weight (w / w). In some embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.01 % to 1 % polysorbate 85 weight to weight (w / w). In other embodiments, the final concentration of polysorbate 85 in the formulation is 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1 % polysorbate 85 (w / w). In another embodiment, the final concentration of the polysorbate 85 in the formulation is 1 % polysorbate 85 (w / w).

[0256] In certain embodiments, the immunogenic composition of the disclosure has a pH of 5.5 to 7.5, for example a pH of 5.6 to 7.0. In a particular embodiment, the immunogenic composition of the disclosure has a pH of 5.8 to 6.0.

[0257] In one embodiment, the present disclosure provides a container filled with any of the immunogenic compositions disclosed herein. In one embodiment, the container is selected from the group consisting of a vial, a syringe, a flask, a fermentor, a bioreactor, a bag, a jar, an ampoule, a cartridge and a disposable pen. In certain embodiments, the container is siliconized.

[0258] In an embodiment, the container of the present disclosure is made of glass, metals (e.g., steel, stainless steel, aluminum, etc.) and / or polymers (e.g., thermoplastics, elastomers, thermoplastic-elastomers). In an embodiment, the container of the present disclosure is made of glass.

[0259] In one embodiment, the present disclosure provides a syringe filled with any of the immunogenic compositions disclosed herein. In certain embodiments, the syringe is siliconized and / or is made of glass.

[0260] A typical dose of the immunogenic composition of the disclosure for injection has a volume of 0.1 mL to 2 mL. In an embodiment, the immunogenic composition of the disclosure for injection has a volume of 0.2 mL to 1 mL. In a particular embodiment, the immunogenic composition of the disclosure for injection has a volume of about 0.5 mL.

[0261] Uses of the Immunogenic Compositions of the Disclosure

[0262] The capsular polysaccharides (CPS) disclosed herein may be used as antigens. For example, they may be part of a vaccine.

[0263] Therefore, in an embodiment, the immunogenic compositions comprising CPS of the disclosure are for use as a medicament. In another embodiment, the immunogenic compositions comprising glycoconjugates of the disclosure are for use as a medicament.

[0264] In an embodiment, the immunogenic compositions comprising CPS of the disclosure are for use as a vaccine. In another embodiment, the immunogenic compositions comprising glycoconjugates of the disclosure are for use as a vaccine.

[0265] Therefore, in an embodiment, the immunogenic compositions described herein are for use in generating an immune response in a subject. In one aspect, the subject is a mammal, such as a human, non-human primate, cat, sheep, pig, horse, bovine or dog. In a particular embodiment, the subject is a human.

[0266] The immunogenic compositions described herein may be used in therapeutic or prophylactic methods for preventing, treating or ameliorating a bacterial infection, disease or condition in a subject. In particular, immunogenic compositions described herein may be used to prevent, treat or ameliorate a S. pneumoniae infection, disease or condition in a subject. For example, the immunogenic compositions described herein may be used to prevent, treat or ameliorate S. pneumoniae infection, disease or condition by the serotypes contained in the composition in a subject.

[0267] Thus, in one aspect, the disclosure provides a method of preventing, treating or ameliorating an infection, disease or condition associated with S. pneumoniae serotypes 15A, 23A, 23B, 24F, and / or 35B in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the disclosure. Thus, in another aspect, the disclosure provides a method of preventing, treating or ameliorating an infection, disease or condition associated with S. pneumoniae serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and / or 35B in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the disclosure.

[0268] In some such embodiments, the infection, disease or condition is selected from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, pleural empyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, and brain abscess.

[0269] In one embodiment, the disclosure provides a method of inducing an immune response to S. pneumoniae serotype 15A, 23A, 23B, 24F, and / or 35B in a subject comprising administering to the subject an immunologically effective amount of an immunogenic composition of the disclosure. In another embodiment, the disclosure provides a method of inducing an immune response to S. pneumoniae serotype 1 , 3,

[0270] 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and / or 35B in a subject comprising administering to the subject an immunologically effective amount of an immunogenic composition of the disclosure.

[0271] In an embodiment, the immunogenic compositions disclosed herein are for use as a vaccine. In one such embodiment the immunogenic compositions described herein may be used to prevent S. pneumoniae serotype 15A, 23A, 23B, 24F, and / or 35B infection in a subject. Thus, in one aspect, the disclosure provides a method of preventing an infection by S. pneumoniae serotype 15A, 23A, 23B, 24F, and / or 35B in a subject comprising administering to the subject an immunologically effective amount of an immunogenic composition of the disclosure. In another such embodiment the immunogenic compositions described herein may be used to prevent

[0272] 5. pneumoniae serotype 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and / or 35B infection in a subject. Thus, in one aspect, the disclosure provides a method of preventing an infection by S. pneumoniae serotype 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and / or 35B in a subject comprising administering to the subject an immunologically effective amount of an immunogenic composition of the disclosure. In some such embodiments, the infection is selected from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, pleural empyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, and brain abscess.

[0273] EXAMPLES

[0274] The following examples demonstrate some embodiments of the present disclosure. However, it is to be understood that these examples are for illustration only and do not purport to be wholly definitive as to conditions and scope of this disclosure. It should be appreciated that when typical reaction conditions (e.g., temperature, reaction times, etc.) have been given, the conditions both above and below the specified ranges can also be used, though generally less conveniently.

[0275] Furthermore, the following examples were carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. As noted above, the following examples are presented for illustrative purpose, and should not be construed in any way limiting the scope of this disclosure.

[0276] Example 1 : Fermentation Optimization- Medium

[0277] The objective of the following examples was to create a simple and robust platform fermentation process to facilitate capsular polysaccharide (CPS) production for serotypes 15A, 23A, 23B, 24F and 35B for use in a Pneumococcal vaccine. A fermentation process was developed to meet the criteria of >1 g / L CPS titer within 24 hours of fermentation time.

[0278] The developed fermentation process leverages an optimized defined medium and a new pressure control strategy. The use of this fermentation process has been demonstrated for Streptococcus pneumoniae strains of serotype 15A, 23A, 23B, 24F, and 35B (the 'additional Pneumococcal vaccine serotypes').

[0279] In order to develop the optimized medium, the additional Pneumococcal vaccine serotypes were evaluated in laboratory fermentation for polysaccharide yield and cell density. Based on these experiments, an optimized medium comprising calcium chloride and potassium phosphate was designed. As shown in Table 2 below, defined media with various concentrations of potassium phosphate and calcium chloride were evaluated for polysaccharide titer and total fermentation time (FT). Each defined medium also included L-Tyrosine disodium dihydrate at a concentration of about 0.643 g / L, D-glucose at a concentration of about 55 g / L, L-asparagine monohydrate at a concentration of about 2.25 g / L, sodium chloride at a concentration of about 1.1 g / L, L-cysteine HCI monohydrate at a concentration of about 0.2504 g / L, L-glutamine at a concentration of about 1.17 g / L, iron(ll) sulfate heptahydrate at a concentration of about 0.00417 g / L, as well as other components. Inclusion of potassium phosphate and calcium chloride in the defined medium resulted in an increase of polysaccharide yield and cell density, as shown in Table 2 below. With the combination of these two supplements in the defined medium, serotype 35B demonstrated a 34% and 39% improvement in polysaccharide yield and cell density, respectively. For serotype 15A, the improvement was 36% and 26% in polysaccharide yield and cell density, respectively.

[0280] Table 2 Fermentation Performance of Pneumococcal Serotypes in a Potassium Phosphate and Calcium Chloride Supplemented Defined Medium Next, the potassium phosphate and calcium chloride supplemented defined medium described above was compared to complex medium (HY-SOY® medium, Sigma-Aldrich®). As shown in Table 3 below, fermentation using potassium phosphate and calcium chloride supplemented defined medium demonstrated a significant improvement in polysaccharide titer (g / L) and polysaccharide specific productivity (g / L / OD) versus fermentation using complex medium. The three serotypes (15A, 23A, and 35B) presented in Table 3 had a significant increase in polysaccharide specific productivity which allowed for an increase in polysaccharide yield.

[0281] Table 3: Fermentation Performance of Pneumococcal Serotypes in a Potassium Phosphate and Calcium Chloride Supplemented Defined Medium

[0282] Example 2: Fermentation Optimization- Backpressure Control Scheme

[0283] An experiment was designed to determine if pressurized fermentation would improve overall fermentation not only by shortening the lag phase, but also shortening the overall fermentation time. S. pneumoniae serotype 35B and 15A strains were used for this comparison study shown in Table 4, below.

[0284] These results demonstrated that the optimized process using a pressurized tank at 5 PSI resulted in a great improvement on shortening fermentation lag phase time and total fermentation time. In fermentation studies with serotype 15A, the pressurized tank resulted in a lag phase that was shortened by more than one hour and a total fermentation time that was shortened by 10 hours as compared to nitrogen overlay only. Furthermore, for the serotype 35B strain, the pressurized tank resulted in a lag phase that was shortened by 4 hours and a total fermentation time that was shortened by 2 hours as compared to nitrogen overlay only. Table 4: Fermentation Backpressure Study Using S. pneumoniae Strain Expressing Serotypes 15A and 35B Next, an S. pneumoniae strain expressing serotype 2 was used for comparison studies. As shown in Table 5 below, the use of a pressurized tank at 5 PSI demonstrated two-fold improvements in shortening fermentation lag phase and increasing specific productivity (g / L / OD / t) as compared to nitrogen overlay in an unpressurized tank. On average, the bacterial lag phase in the pressurized tanks was shortened by more than 50% compared with nitrogen overlay tanks (from 21 hrs. down to 10 hrs.). The polysaccharide yield (g / L) in the pressurized tank also increased from 3.4 g / L to 4.7 g / L.

[0285] Table 5: Fermentation Back Pressure Study Using S. Pneumoniae Strain Expressing Serotype 2

[0286] Accordingly, this Example demonstrates that fermentation in a pressurized tank is a viable method to decrease fermentation time, increase polysaccharide titer, and increase specific productivity.

[0287] Example 3: Fermentation Parameters

[0288] A fermentation process according to the disclosure for the additional Pneumococcal vaccine serotypes incorporated the medium described in Example 1 and the backpressure control scheme (5 PSI) described in Example 2. The final process was tested in batches at 75L scale for all five serotypes.

[0289] The medium components were sterile filtered into a respective fermenter and adjusted to a designated volume. All post sterile additions, including 1 M sodium bicarbonate and 20% w / v magnesium sulfate heptahydrate, were added prior to inoculation.

[0290] The fermentation operating conditions are shown in Table 6 below.

[0291] Table 6: Fermentation Operation Parameters Immediately post inoculation, the initial glucose concentration was determined. The subsequent hourly samples were collected after 10-16 hours of fermentation. Cell density was determined by optical density measures for absorbance at 600 nm (OD600) with appropriate dilution to maintain spectrophotometer readings of <0.5. In process samples of 5 mL were taken for determination of polysaccharide titer. These samples were lysed with 0.1 % v / v N-lauroyl-sarcosine (NLS), incubated at 36°C for about 1 hour, and then held at 4° C overnight prior to sample preparation for high performance liquid chromatography (HPLC) analysis.

[0292] The batches were considered finished when the OD600 reading increased by <0.5 over the course of an hour. At this time, the pH controller was turned off and the batch was immediately lysed by the addition of 0.1 % v / v NLS. The batches are then held at 36° C for 30 minutes with stirring. The batches were then ready for downstream processing.

[0293] The final process was tested in batches at 75L scale for all five serotypes. The average fermentation time, average cell density, and average polysaccharide titer for each of the serotypes is shown in Table 7 below.

[0294] Table 7: Fermentation at 75L Scale

[0295] The results presented in Table 7 clearly show that the tested fermentation process demonstrated consistency in polysaccharide yield and growth rate for each of the five serotypes. Based on these results, the fermentation process can be scaled up to 2000 L.

[0296] In this study, a fermentation process was developed to meet the criteria of >1 g / L CPS titer within 24 hours of fermentation time. In order to achieve these criteria for all five serotypes, several fermentation process improvements were identified and implemented. These improvements included optimizing the defined medium components and optimizing the backpressure. The following clauses describe additional embodiments of the disclosure:

[0297] C1 . A medium for use in culturing a bacterial cell culture, comprising a compound comprising potassium and a compound comprising calcium, wherein the bacterial cell culture produces capsular polysaccharides.

[0298] C2. The medium of C1 , wherein the compound comprising potassium comprises potassium phosphate.

[0299] C3. The medium of C2, wherein the concentration of the potassium phosphate is between about 0.1 grams per liter of medium (g / L) and about 10 g / L.

[0300] C4. The medium of C2 or C3, wherein the concentration of the potassium phosphate is between about 0.5 g / L and about 2 g / L.

[0301] C5. The medium of any one of C2-C4, wherein the concentration of the potassium phosphate is about 1 g / L.

[0302] C6. The medium of any one of C2-C5, wherein the potassium phosphate is monobasic potassium phosphate.

[0303] C7. The medium of any one of C1 -C6, wherein the compound comprising calcium comprises calcium chloride.

[0304] C8. The medium of C7, wherein the concentration of the calcium chloride is between about 0.01 g / L and about 10 g / L.

[0305] C9. The medium of C7 or C8, wherein the concentration of the calcium chloride is between about 0.1 g / L and about 1 g / L.

[0306] C10. The medium of any one of C7-C9, wherein the concentration of the calcium chloride is between about 0.1 g / L and about 0.2 g / L. C11 . The medium of any one of C7-C10, wherein the concentration of the calcium chloride is about 0.1 g / L, about 0.11 g / L, about 0.12 g / L, about 0.13 g / L, about 0.14 g / L, about 0.15 g / L, about 0.16 g / L, about 0.17 g / L, about 0.18 g / L, about 0.19, or about 0.2 g / L.

[0307] C12. The medium of any one of C7-C11 , wherein the concentration of the calcium chloride is between about 0.13 g / L and about 0.14 g / L.

[0308] C13. The medium of any one of C7-C12, wherein the concentration of the calcium chloride is about 0.13 g / L.

[0309] C14. The medium of any one of C7-C13, wherein the concentration of the calcium chloride is about 0.13374 g / L.

[0310] C15. The medium of any one of C7-C14, wherein the calcium chloride is anhydrous.

[0311] C16. The medium of any one of C1 -C15, wherein the medium further comprises tyrosine.

[0312] C17. The medium of C16, wherein the tyrosine is L-tyrosine.

[0313] C18. The medium of C17, wherein the L-Tyrosine comprises L-Tyrosine disodium di hydrate.

[0314] C19. The medium of C18, wherein the concentration of the L-Tyrosine disodium dihydrate is between about 0.1 g / L and about 1 g / L.

[0315] C20. The medium of C18 or C19, wherein the concentration of the L-Tyrosine disodium dihydrate is between about 0.6 g / L and about 0.7 g / L.

[0316] C21 . The medium of any one of C18-C20, wherein the concentration of the L- Tyrosine disodium dihydrate is about 0.64 g / L. C22. The medium of any one of C18-C21 , wherein the concentration of the L- Tyrosine disodium dihydrate is about 0.643 g / L.

[0317] C23. The medium of any one of C1 -C22, further comprising glucose.

[0318] C24. The medium of C23, wherein the glucose is D-glucose.

[0319] C25. The medium of C23 or C24, wherein the concentration of the glucose is between about 10 g / L and about 100 g / L.

[0320] C26. The medium of any one of C23-C25, wherein the concentration of the glucose is between about 40 g / L and about 60 g / L.

[0321] C27. The medium of any one of C23-C26, wherein the concentration of the glucose is between about 50 g / L and about 60 g / L.

[0322] C28. The medium of any one of C23-C27, wherein the concentration of the glucose is about 55 g / L.

[0323] C29. The medium of any one of C1 -C28, further comprising asparagine.

[0324] C30. The medium of C29, wherein the asparagine is L-asparagine.

[0325] C31 . The medium of C30, wherein the L-asparagine comprises L-asparagine monohydrate.

[0326] C32. The medium of C31 , wherein the concentration of the L-asparagine monohydrate is between about 0.5 g / L and about 10 g / L.

[0327] C33. The medium of C31 or C32, wherein the concentration of the L-asparagine monohydrate is between about 1 g / L and about 5 g / L. C34. The medium of any one of C31 -C33, wherein the concentration of the L- asparagine monohydrate is between about 2 g / L and about 3 g / L.

[0328] C35. The medium of any one of C31 -C34, wherein the concentration of the L- asparagine monohydrate is about 2.25 g / L.

[0329] C36. The medium of any one of C1 -C35, further comprising sodium chloride.

[0330] C37. The medium of C36, wherein the concentration of the sodium chloride is between about 0.5 g / L and about 10 g / L.

[0331] C38. The medium of C36 or 37, wherein the concentration of the sodium chloride is between about 0.5 g / L and about 1 .5 g / L.

[0332] C39. The medium of any one of C36-C38, wherein the concentration of the sodium chloride is about 1.1 g / L.

[0333] C40. The medium of any one of C1 -C39, further comprising cysteine.

[0334] C41 . The medium of C40, wherein the cysteine is L-cysteine.

[0335] C42. The medium of C41 , wherein the L-cysteine comprises L-cysteine HCI monohydrate.

[0336] C43. The medium of C42, wherein the concentration of the L-cysteine HCI monohydrate is between about 0.1 g / L and about 10 g / L.

[0337] C44. The medium of C42 or C43, wherein the concentration of the L-cysteine HCI monohydrate is between about 0.1 g / L and about 1 g / L.

[0338] C45. The medium of any one of C42-C44, wherein the concentration of the L- cysteine HCI monohydrate is between about 0.2 g / L and about 0.3 g / L. C46. The medium of any one of C42-C45, wherein the concentration of the L- cysteine HCI monohydrate is about 0.25 g / L.

[0339] C47. The medium of any one of C42-C46, wherein the concentration of the L- cysteine HCI monohydrate is about 0.2504 g / L.

[0340] C48. The medium of any one of C1 -C47, further comprising glutamine.

[0341] C49. The medium of C48, wherein the glutamine is L-glutamine.

[0342] C50. The medium of C49, wherein the concentration of the L-glutamine is between about 0.5 g / L and about 10 g / L.

[0343] C51 . The medium of C49 or C50, wherein the concentration of the L-glutamine is between about 0.5 g / L and about 2.5 g / L.

[0344] C52. The medium of any one of C49-C51 , wherein the concentration of the L- glutamine is between about 1 g / L and about 2 g / L.

[0345] C53. The medium of any one of C49-C52, wherein the concentration of the L- glutamine is between about 1.1 g / L and about 1 .2 g / L.

[0346] C54. The medium of any one of C49-C53, wherein the concentration of the L- glutamine is about 1.17 g / L.

[0347] C55. The medium of any one of C1 -C54, wherein the medium further comprises iron sulfate.

[0348] C56. The medium of C55, wherein the iron sulfate comprises Iron(ll) sulfate heptahydrate.

[0349] C57. The medium of C56, wherein the concentration of Iron(ll) sulfate heptahydrate is between about 0.00001 g / L and about 0.01 g / L. C58. The medium of C56 or C57, wherein the concentration of Iron(ll) sulfate heptahydrate is between about 0.001 g / L and about 0.01 g / L.

[0350] C59. The medium of any one of C56-C58, wherein the concentration of Iron(ll) sulfate heptahydrate is between about 0.004 g / L and about 0.005 g / L.

[0351] C60. The medium of any one of C56-C59, wherein the concentration of Iron(ll) sulfate heptahydrate is about 0.004 g / L.

[0352] C61 . The medium of any one of C56-C60, wherein the concentration of Iron(ll) sulfate heptahydrate is about 0.00417 g / L.

[0353] C62. The medium of any one of C1 -C61 , wherein the medium comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 of the medium components provided in Table 1 .

[0354] C63. The medium of any one of C1 -C62, wherein the medium consists of calcium chloride, potassium phosphate, and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 of the medium components provided in Table 1 .

[0355] C64. The medium of any one of C1 -C63, wherein the medium consists of calcium chloride, potassium phosphate, and each of the medium components provided in Table 1 .

[0356] C65. The medium of any one of C1 -C64, wherein the medium is a pneumococcal growth medium.

[0357] C66. The medium of any one of C1 -C65, wherein the medium is a defined pneumococcal growth medium. C67. A method of increasing the capsular polysaccharide titer measured in grams per liter (g / L) produced by a bacterial cell culture over a unit of time, comprising culturing the bacterial cell culture in the medium of any one of C1-C66.

[0358] C68. The method of C67, wherein the unit of time is between about 1 hour and about 72 hours.

[0359] C69. The method of C67 or C68, wherein the unit of time is about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, or about 32 hours.

[0360] C70. The method of any one of C67-C69, wherein the unit of time is about 24 hours.

[0361] C71 . The method of any one of C67-C70, wherein the polysaccharide titer is between about 0.1 g / L and about 10 g / L.

[0362] C72. The method of any one of C67-C71 , wherein the polysaccharide titer is at least about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 1.1 g / L, about 1 .2 g / L, about 1 .3 g / L, about 1 .4 g / L, about 1 .5 g / L, about 1.6 g / L, about 1 .7 g / L, about 1 .8 g / L, about 1 .9 g / L, about 2 g / L, about 2.1 g / L, about 2.2 g / L, about 2.3 g / L, about 2.4 g / L, or about 2.5 g / L.

[0363] C73. The method of any one of C67-C72, wherein the polysaccharide titer is at least about 1 g / L or about 2 g / L.

[0364] C74. The method of any one of C67-C73, wherein the polysaccharide titer is at least 1 g / L or about 2 g / L, and the unit of time is about 24 hours.

[0365] C75. A method of increasing capsular polysaccharide specific productivity measured in grams polysaccharide per liter per OD (g / L / OD) of a bacterial cell culture over a unit of time, comprising culturing the bacterial cell culture in the medium of any one of C1 -C66. C76. The method of C75, wherein the unit of time is between about 1 hour and about 72 hours.

[0366] C77. The method of C75 or C76, wherein the unit of time is about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, or about 32 hours.

[0367] C78. The method of any one of C75-C77, wherein the unit of time is about 24 hours.

[0368] C79. The method of any one of C75-C78, wherein the polysaccharide specific productivity is between about 0.05 g / L / OD and about 0.5 g / L / OD.

[0369] C80. The method of any one of C75-C79, wherein the polysaccharide specific productivity is between about 0.1 g / L / OD and about 0.3 g / L / OD.

[0370] C81 . The method of any one of C75-C80, wherein the polysaccharide specific productivity is about 0.1 , about 0.11 , about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.2, about 0.21 , about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, or about 0.3 g / L / OD.

[0371] C82. A method of culturing a bacterial cell culture in a bioreactor with backpressure, wherein the bacterial cell culture produces capsular polysaccharides.

[0372] C83. The method of any one of C67-C81 , further comprising culturing the bacterial cell culture in a bioreactor with backpressure.

[0373] C84. The method of C82 or C83, wherein the backpressure is between about 1 PSI and about 20 PSI. C85. The method of any one of C82-C84, wherein the backpressure is about 1 PSI, about 2 PSI, about 3 PSI, about 4 PSI, or about 5 PSI.

[0374] C86. The method of any one of C82-C85, wherein the backpressure is about 5 PSI.

[0375] C87. The method of any one of C82-C86, wherein the lag phase of the bacteria in the bacterial cell culture is decreased as compared to the lag phase of bacteria in a bacterial cell culture cultured in a bioreactor without backpressure.

[0376] C88. The method of C87, wherein the lag phase of the bacteria is decreased by between about 0.5 hours and about 15 hours.

[0377] C89. The method of C87 or C88, wherein the lag phase of the bacteria is decreased by about 0.5, about 1 , about 1 .5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11 , about 11.5, or about 12 hours.

[0378] C90. The method of any one of C87-C89, wherein the lag phase of the bacteria is decreased by about 1 hour.

[0379] C91 . The method of any one of C87-C89, wherein the lag phase of the bacteria is decreased by about 4 hours.

[0380] C92. The method of any one of C82-C91 , wherein the total culturing time required to achieve a target capsular polysaccharide yield is decreased as compared to the total culturing time required to achieve the target capsular polysaccharide yield with a bacterial cell culture cultured in a bioreactor without backpressure.

[0381] C93. The method of C92, wherein the total culturing time is decreased by between about 0.5 hours and about 15 hours.

[0382] C94. The method of C92 or C93, wherein the total culturing time is decreased by about 0.5, about 1 , about 1 .5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11 , about 11.5, or about 12 hours.

[0383] C95. The method of any one of C92-C94, wherein the total culturing time is decreased by about 2 hours.

[0384] C96. The method of any one of C92-C94, wherein the total culturing time is decreased by about 10 hours.

[0385] C97. The method of any one of C92-C96, wherein the target polysaccharide yield is between about 1 gram per liter (g / L) and about 5 g / L.

[0386] C98. The method of any one of C92-C97, wherein the target polysaccharide titer is about 1 g / L, about 1.5 g / L, about 1 .6 g / L, about 1 .7 g / L, about 1 .8 g / L, about 1 .9 g / L, or about 2 g / L.

[0387] C99. The method of any one of C82-C98, wherein the method does not comprise the use of nitrogen overlay.

[0388] C100. The method of any one of C82-C98, wherein the method further comprises the use of nitrogen overlay.

[0389] C101 . The method of any one of C82 or C84-C100, wherein the bacterial cell culture is cultured in the medium of any one of C1-C66.

[0390] C102. The medium or method of any one of C1 -C101 , wherein the bacteria in the bacterial cell culture is selected from the group consisting of Streptococcus agalactiae, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria meningitidis, Escherichia coli, Salmonella typhi, Haemophilus influenzae, Klebsiella pneumoniae, Enterococcus faecium, and Enterococcus faecalis.

[0391] C103. The medium or method of any one of C1 -C102, wherein the bacteria in the bacterial cell culture is Streptococcus pneumoniae. C104. The medium or method of C103, wherein the serotype of the Streptococcus pneumoniae is 15A.

[0392] C105. The medium or method of C103, wherein the serotype of the Streptococcus pneumoniae is 23A.

[0393] C106. The medium or method of C103, wherein the serotype of the Streptococcus pneumoniae is 23B.

[0394] C107. The medium or method of C103, wherein the serotype of the Streptococcus pneumoniae is 24F.

[0395] C108. The medium or method of C103, wherein the serotype of the Streptococcus pneumoniae is 35B.

[0396] C109. A capsular polysaccharide produced by the medium or method of any one of C1-C108.

[0397] C110. An immunogenic composition comprising the capsular polysaccharide of C109.

[0398] C111 . The immunogenic composition of C110, wherein the immunogenic composition comprises a capsular polysaccharide from a serotype selected from the group consisting of Streptococcus pneumoniae serotypes 15A, 23A, 23B, 24F, and 35B.

[0399] C112. The immunogenic composition of C110 or C111 , wherein the immunogenic composition comprises capsular polysaccharides from each of Streptococcus pneumoniae serotypes 15A, 23A, 23B, 24F, and 35B.

[0400] C113. The immunogenic composition of any one of C110-C112, wherein the immunogenic composition further comprises capsular polysaccharides from at least one of the Streptococcus pneumoniae serotypes selected from the group consisting of 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

[0401] C114. The immunogenic composition of any one of C110-C113, wherein the immunogenic composition further comprises capsular polysaccharides from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, or 19 of the Streptococcus pneumoniae serotypes selected from the group consisting of 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

[0402] C115. The immunogenic composition of any one of C110-C114, wherein the immunogenic composition comprises capsular polysaccharides from each of Streptococcus pneumoniae serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B.

[0403] C116. The immunogenic composition of any one of C110-C115, wherein the immunogenic composition consists of the capsular polysaccharides from each of Streptococcus pneumoniae serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B.

[0404] C117. The immunogenic composition of any one of C110-C116, wherein at least one of the capsular polysaccharides is conjugated to a carrier protein to form a glycoconjugate.

[0405] C118. The immunogenic composition of any one of C110-C117, wherein at least one of the capsular polysaccharides is conjugated to CRMigyto form a glycoconjugate.

[0406] C119. The immunogenic composition of any one of C110-C118, wherein each of the capsular polysaccharides are conjugated to CRMigyto form a glycoconjugate.

[0407] C120. The immunogenic composition of any one of C110-C118, wherein at least one of the capsular polysaccharides are conjugated to SCP to form a glycoconjugate. C121 . The immunogenic composition of any one of C110-C117 or C120, wherein each of the capsular polysaccharides are conjugated to SCP to form a glycoconjugate.

[0408] C122. The immunogenic composition of any one of C113-C118 or C120, wherein the capsular polysaccharide from S. pneumoniae serotype 3 is conjugated to SCP and the other capsular polysaccharides are all individually conjugated to CRM197.

[0409] C123. A method of inducing an immune response to S. pneumoniae serotypes 15A, 23A, 23B, 24F, and / or 35B in a subject comprising administering the immunogenic composition of any one of C110-C122 to the subject.

[0410] C124. A method of inducing an immune response to S. pneumoniae serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and / or 35B in a subject comprising administering the immunogenic composition of any one of C113-C122 to the subject.

[0411] C125. Use of the immunogenic composition of any one of C110-C122, for inducing an immune response to S. pneumoniae serotypes 15A, 23A, 23B, 24F, and / or 35B in a subject.

[0412] C126. Use of the immunogenic composition of any one of C113-C122, for inducing an immune response to S. 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 / or 35B in a subject.

[0413] C127. Use of the immunogenic composition of any one of C110-C122, in the manufacture of a medicament for inducing an immune response to S. pneumoniae serotypes 15A, 23A, 23B, 24F, and / or 35B in a subject.

[0414] C128. Use of the immunogenic composition of any one of C113-C122, in the manufacture of a medicament for inducing an immune response to S. pneumoniae serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and / or 35B in a subject.

Claims

CLAIMS:1 . A medium for use in culturing a bacterial cell culture, comprising potassium phosphate and calcium chloride, wherein the bacterial cell culture produces capsular polysaccharides.

2. The medium of claim 1 , wherein the concentration of the potassium phosphate is between about 0.5 grams per liter of medium (g / L) and about 2 g / L.

3. The medium of claim 1 or 2, wherein the concentration of the potassium phosphate is about 1 g / L.

4. The medium of any one of claims 1 -3, wherein the potassium phosphate is monobasic potassium phosphate.

5. The medium of any one of claims 1 -4, wherein the concentration of the calcium chloride is between about 0.1 g / L and about 0.2 g / L.

6. The medium of any one of claims 1 -5, wherein the concentration of the calcium chloride is between about 0.13 g / L and about 0.14 g / L.

7. The medium of any one of claims 1 -6, wherein the concentration of the calcium chloride is about 0.13374 g / L.

8. The medium of any one of claims 1 -7, wherein the calcium chloride is anhydrous.

9. The medium of any one of claims 1 -8, wherein the medium further comprises glucose.

10. The medium of claim 9, wherein the glucose has a concentration between about 50 g / L and about 60 g / L.11 . The medium of any one of claims 1 -10, wherein the medium further comprises a component selected from the group consisting of tyrosine, asparagine, sodium chloride, cysteine, glutamine, and iron sulfate.

12. The medium of any one of claims 1-11 , wherein the composition further comprises each of the following components: tyrosine, asparagine, sodium chloride, cysteine, glutamine, and iron sulfate.

13. The medium of claim 11 or 12, wherein the tyrosine comprises L-Tyrosine disodium dihydrate and the concentration of the L-Tyrosine disodium dihydrate is between about 0.1 g / L and about 1 g / L.

14. The medium of any one of claims 11-13, wherein the asparagine comprises L- asparagine monohydrate and the concentration of the L-asparagine monohydrate is between about 2 g / L and about 3 g / L.

15. The medium of any one of claims 11-14, wherein the concentration of the sodium chloride is between about 0.5 g / L and about 1 .5 g / L.

16. The medium of any one of claims 11-15, wherein the cysteine comprises L- cysteine HCI monohydrate and the concentration of the L-cysteine HCI monohydrate is between about 0.1 g / L and about 1 g / L.

17. The medium of any one of claims 11-16, wherein the glutamine is L-glutamine and the concentration of the L-glutamine is between about 1 g / L and about 2 g / L.

18. The medium of any one of claims 11-17, wherein the iron sulfate comprises Iron(ll) sulfate heptahydrate and the concentration of Iron(ll) sulfate heptahydrate is between about 0.001 g / L and about 0.01 g / L.

19. The medium of any one of claims 1 -18, wherein the medium comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46,47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 of the medium components provided in Table 1.

20. The medium of any one of claims 1 -19, wherein the medium consists of calcium chloride, potassium phosphate, and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 of the medium components provided in Table 1.21 . The medium of any one of claims 1 -20, wherein the medium consists of calcium chloride, potassium phosphate, and each of the medium components provided in Table 1.

22. The medium of any one of claims 1 -21 , wherein the medium is a pneumococcal growth medium.

23. The medium of any one of claims 1 -22, wherein the medium is a defined pneumococcal growth medium.

24. A method of increasing the capsular polysaccharide titer measured in grams per liter (g / L) produced by a bacterial cell culture over a unit of time, comprising culturing the bacterial cell culture in the medium of any one of claims 1 -23.

25. The method of claim 24, wherein the unit of time is about 24 hours.

26. The method of claim 24 or 25, wherein the polysaccharide titer is at least about 1 g / L or at least about 2 g / L.

27. A method of increasing capsular polysaccharide specific productivity measured in grams polysaccharide per liter per OD (g / L / OD) of a bacterial cell culture over a unit of time, comprising culturing the bacterial cell culture in the medium of any one of claims 1 -23.

28. The method of claim 27, wherein the unit of time is about 24 hours.

29. The method of claim 27 or 28, wherein the polysaccharide specific productivity is between about 0.1 g / L / OD and about 0.3 g / L / OD.

30. The method of any one of claims 24-29, further comprising culturing the bacterial cell culture in a bioreactor wherein the backpressure is about 1 PSI, about 2 PSI, about 3 PSI, about 4 PSI, or about 5 PSI.31 . A method of culturing a bacterial cell culture in a bioreactor wherein the backpressure is about 1 PSI, about 2 PSI, about 3 PSI, about 4 PSI, or about 5 PSI, and wherein the bacterial cell culture produces capsular polysaccharides.

32. The method of claim 30 or 31 , wherein the backpressure is about 5 PSI.

33. The method of any one of claims 30-32, wherein the lag phase of the bacteria in the bacterial cell culture is decreased as compared to the lag phase of bacteria in a bacterial cell culture cultured in a bioreactor without backpressure.

34. The method of claim 33, wherein the lag phase of the bacteria is decreased by between about 0.5 hours and about 5 hours.

35. The method of any one of claims 30-34, wherein the total culturing time required to achieve a target capsular polysaccharide yield is decreased as compared to the total culturing time required to achieve the target capsular polysaccharide yield with a bacterial cell culture cultured in a bioreactor without backpressure.

36. The method of claim 35, wherein the total culturing time is decreased by between about 1 hours and about 10 hours.

37. The method of claim 35 or 36, wherein the target polysaccharide yield is about 1 g / L, about 1 .5 g / L, about 1 .6 g / L, about 1 .7 g / L, about 1 .8 g / L, about 1 .9 g / L, or about 2 g / L.

38. The method of any one of claims 30-37, wherein the method does not comprise the use of nitrogen overlay.

39. The method of any one of claims 30-38, wherein the bacterial cell culture is cultured in the medium of any one of claims 1-23.

40. The medium or method of any one of claims 1-39, wherein the bacteria in the bacterial cell culture is selected from the group consisting of Streptococcus agalactiae, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria meningitidis, Escherichia coli, Salmonella typhi, Haemophilus influenzae, Klebsiella pneumoniae, Enterococcus faecium, and Enterococcus faecalis.41 . The medium or method of claim 40, wherein the bacteria in the bacterial cell culture is Streptococcus pneumoniae.

42. The medium or method of claim 41 , wherein the serotype of the Streptococcus pneumoniae is selected from the group consisting of 15A, 23A, 23B, 24F, and 35B.

43. A capsular polysaccharide produced by the medium or method of any one of claims 1-42.

44. An immunogenic composition comprising the capsular polysaccharide of claim 43.

45. The immunogenic composition of claim 44, wherein the immunogenic composition comprises capsular polysaccharides from a serotype selected from the group consisting of Streptococcus pneumoniae serotypes 15A, 23A, 23B, 24F, and 35B.

46. The immunogenic composition of claim 44 or 45, wherein the immunogenic composition comprises capsular polysaccharides from each of Streptococcus pneumoniae serotypes 15A, 23A, 23B, 24F, and 35B, and wherein each of thecapsular polysaccharides are produced by the medium or method of any one of claims 1-42.

47. A method of inducing an immune response to Streptococcus pneumoniae serotypes 15A, 23A, 23B, 24F, and / or 35B in a subject comprising administering the immunogenic composition of claim 45 or 46 to the subject.

48. The immunogenic composition of any one of claims 44-46, wherein the immunogenic composition further comprises capsular polysaccharides from each of the following Streptococcus pneumoniae serotypes: 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

49. A method of inducing an immune response to Streptococcus pneumoniae serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and / or 35B in a subject comprising administering the immunogenic composition of claim 48 to the subject.