Media and fermentation methods for producing polysaccharides in bacterial cell cultures - Patents.com

Abstract

Media and fermentation methods for producing polysaccharides in bacterial cell cultures are provided. The present invention relates to a complex culture medium for culturing Streptococcus pneumoniae or Streptococcus agalactiae, the culture medium comprising a plant hydrolysate, yeast extract, and a carbon source. In another aspect, the present invention relates to a defined medium having a total amino acid concentration greater than about 50 mM. A further aspect of the present invention relates to the use of fed-batch and perfusion fermentation methods for growing polysaccharide-producing bacteria.

Description

[Technical Field]

[0001] The present invention relates to media and fermentation methods for producing polysaccharides in bacterial cell cultures. In one aspect, the invention relates to a complex culture medium comprising a plant hydrolysate, a yeast extract, and a carbon source. In another aspect, the invention relates to a defined medium having a total amino acid concentration greater than about 50 mM. A further aspect of the invention relates to the use of fed-batch and perfusion fermentation methods for growing polysaccharide-producing bacteria. [Background technology]

[0002] Cell surface polysaccharides refer to polysaccharides that are at least partially located on the outermost bacterial cell membrane or cell surface, including the peptidoglycan layer, cell wall, and capsule. Cell surface polysaccharides, particularly capsular polysaccharides, are becoming increasingly important as therapeutic agents. Typically, cell surface polysaccharides are associated with eliciting immune responses in vivo. Some examples of polysaccharide vaccines include PNEUMOVAX® 23, a 23-valent vaccine for the prevention of invasive diseases caused by Streptococcus pneumoniae, such as pneumonia, febrile bacteremia, and meningitis; MENCEVAX®, a tetravalent vaccine for the prevention of invasive diseases caused by Neisseria meningitidis; and TYPHERIX® and TYPHIM VI®, both of which prevent typhoid fever caused by Salmonella typhi Vi.

[0003] Although polysaccharides themselves are immunogenic, conjugation of polysaccharides to protein carriers has been used to improve immunogenicity, especially in infants and the elderly. For bacteria that display polysaccharides on their surface, the chemical bond between the polysaccharide and the protein carrier induces an immune response and thus prevents disease. Therefore, vaccination using polysaccharides derived from pathogenic bacteria may be a strategy for boosting host immunity.

[0004] Several polysaccharide-protein conjugate vaccines are currently available, and several more are in development to address therapeutic areas of unmet need. For example, three pneumococcal conjugate vaccines used to protect against invasive pneumococcal disease are available on the global market: PREVNAR® (also called PREVENAR® in some countries) (7-valent vaccine), SYNFLORIX® (10-valent vaccine), and PREVNAR13® (13-valent vaccine). MENINGITEC®, MENJUGATE®, and NEISVAC-C® are meningococcal serogroup C conjugate vaccines, while MENVEO®, MENACTRA®, and NIMENRIX® are quadrivalent meningococcal conjugate vaccines that protect against N. meningitidis serogroups A, C, Y, and W-135. HIBERIX® prevents disease caused by Haemophilus influenzae type b.

[0005] Monovalent polysaccharide-protein conjugates of Streptococcus agalactiae serotypes Ia, Ib, II, III, and V, also known as group B streptococcus (GBS), have been individually evaluated in phase I and II clinical trials in non-pregnant adults (Brigtsen, A.K. et al., Journal of Infectious Diseases, 185(9):1277-1284 (2002); Baker, C.J. et al., J. Infect. Dis., 188(1):66-73 (2003); Baker, C.J. et al., J. Infect. Dis., 189(6):1103-1112 (2004); Baker, C.J. et al., Vaccine, 25(1):55-63 (2007)). Bivalent glycoconjugate vaccines of II-TT and III-TT, as well as Ia-CRM, have been evaluated in non-pregnant adults (Brigtsen, A.K. et al., Journal of Infectious Diseases, 185(9):1277-1284 (2002); Baker, C.J. et al., J. Infect. Dis., 188(1):66-73 (2003); Baker, C.J. et al., J. Infect. Dis., 189(6):1103-1112 (2004); Baker, C.J. et al., Vaccine, 25(1):55-63 (2007)). 197 , Ib-CRM 197 and III-CRM 197Trivalent vaccines containing glycoconjugates have also been investigated (Baker, JID, 2003; Clinicaltrials.gov NCT01193920, NCT01412801, and NCT01446289), but no GBS vaccine has yet been approved.

[0006] In addition, vaccines containing conjugates of capsular polysaccharides and proteins are being developed to prevent surgical site infections caused by Staphylococcus aureus (Anderson, AS et al., Hum. Vaccin. Immunother., 8(11):1585-1594(2012)). Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, there is a need to develop improved systems for producing polysaccharides in bacterial cell culture. [Means for solving the problem]

[0008] To meet these and other needs, the present invention relates to media and fermentation methods for producing polysaccharides in bacterial cell cultures, including those disclosed in U.S. Provisional Patent Application No. 62 / 256,347, filed November 17, 2015, which is incorporated herein by reference in its entirety. The following sections describe several aspects and embodiments of the invention.

[0009] One aspect of the present invention relates to a medium for culturing polysaccharide-producing bacterial cells, the medium comprising a plant hydrolysate, a yeast extract and a carbon source. In one embodiment, the vegetable hydrolysate may be a soy hydrolysate such as HYPEP1510 (Kerry Group Services Ltd.), HYPEP4601 (Kerry Group Services Ltd.), HYPEP5603 (Kerry Group Services Ltd.), HY-SOY (Kerry Group Services Ltd.), AMI-SOY (Kerry Group Services Ltd.), NZ-SOY (Kerry Group Services Ltd.), NZ-SOY BL4 (Kerry Group Services Ltd.), NZ-SOY BL7 (Kerry Group Services Ltd.), SHEFTONE D (Kerry Group Services Ltd.), SE50M, SE50MK, soy peptone, BACTO Soytone (Difco Laboratories Inc.), NUTRISOY2207 (Archer Daniels Midland Company (ADM)), NUTRISOY (ADM), NUTRISOY FLOUR (ADM), or soybean meal. In another embodiment, the concentration of the soy hydrolysate can be between about 5 g / L and about 75 g / L, for example, between about 10 g / L and about 50 g / L, or about 28 g / L.

[0010] In further embodiments, the yeast extract may be a yeast autolysate, an ultrafiltered yeast extract, or a synthetic yeast extract. In certain embodiments, the yeast extract is an ultrafiltered yeast extract, such as AMBERFERM5902 (Sensient Technologies Corp.), BD DIFCO (BD Biosciences), HYPEP YE (Kerry Group Services Ltd.), HY-YEST412 (Kerry Group Services Ltd.), HY-YEST441 (Kerry Group Services Ltd.), HY-YEST444 (Kerry Group Services Ltd.), HY-YEST455 (Kerry Group Services Ltd.), HY-YEST504 (Kerry Group Services Ltd.), or ULTRAPEP YE (Kerry Group Services Ltd.). In yet other embodiments, the concentration of the yeast extract is between about 1 g / L and about 50 g / L, for example, between about 5 g / L and about 25 g / L, or about 10 g / L.

[0011] In one embodiment, the carbon source can be glucose, dextrose, mannitol, lactose, sucrose, fructose, galactose, raffinose, xylose, or mannose. In a specific embodiment, the carbon source is glucose. In a further embodiment, the concentration of the carbon source is between about 25 g / L and about 100 g / L, e.g., between about 50 g / L and about 90 g / L, or about 80 g / L.

[0012] In one embodiment, the medium further comprises a phosphate-containing component, such as Na2HPO4, K2HPO4, or KH2PO4.

[0013] In another embodiment, the medium further comprises at least one amino acid, vitamin, nucleoside, or inorganic salt.

[0014] Another aspect of the present invention relates to a chemically defined medium for culturing polysaccharide-producing bacterial cells, the medium having a total concentration of amino acids greater than about 50 mM. In one embodiment, the medium contains glycine at a total concentration of between about 1.5 mM and about 60.0 mM, e.g., between about 5.0 mM and about 15.0 mM, or about 7.5 mM. In another embodiment, the medium contains arginine at a total concentration of between about 1.0 mM and about 30.0 mM, e.g., between about 1.0 mM and about 20.0 mM, or about 4.0 mM. In a further embodiment, the medium contains cysteine ​​at a total concentration of between about 0.1 mM and about 5.0 mM, e.g., between about 0.1 mM and about 3.5 mM, or about 0.4 mM. In yet another embodiment, the medium contains serine at a total concentration of about 5.0 mM to about 75.0 mM, for example, about 5.0 mM to about 15.0 mM, or about 7.5 mM. In another embodiment, the medium contains glutamine at a total concentration of about 1.0 mM to about 30.0 mM, for example, about 1.0 mM to about 20.0 mM, or about 4.0 mM. In a further embodiment, the medium contains tyrosine at a total concentration of about 0.1 mM to about 5.0 mM, for example, about 1.0 mM to about 3.5 mM, or about 2.9 mM to about 3.0 mM. In yet another embodiment, the medium contains asparagine at a total concentration of about 5.0 mM to about 50.0 mM, for example, about 10.0 mM to about 30.0 mM, or about 20.0 mM. In a specific embodiment, the medium does not contain asparagine.

[0015] In one embodiment, the medium further comprises a potassium salt, such as potassium chloride or potassium sulfate, in which the total concentration of the potassium salt is between about 0.1 g / L and about 25 g / L, e.g., between about 0.2 g / L and about 1.25 g / L, or about 0.9 g / L.

[0016] In one embodiment, the medium further comprises a carbon source, such as glucose, dextrose, mannitol, lactose, sucrose, fructose, galactose, raffinose, xylose, or mannose. In a specific embodiment, the carbon source is glucose. In certain embodiments, the total concentration of the carbon source can be between about 25 g / L and about 100 g / L, for example, between about 25 g / L and about 80 g / L, or about 50 g / L.

[0017] In one embodiment, the medium further comprises sodium bicarbonate, which may have a concentration of between about 0.1 g / L and about 20 g / L, e.g., between about 0.5 g / L and about 1.0 g / L, or about 0.84 g / L.

[0018] In one embodiment, the medium further comprises a yeast extract, such as a yeast autolysate, an ultrafiltered yeast extract, or a synthetic yeast extract. In a specific embodiment, the yeast extract is an ultrafiltered yeast extract, such as AMBERFERM5902 (Sensient Technologies Corp.), BD DIFCO (BD Biosciences), HYPEP YE (Kerry Group Services Ltd.), HY-YEST412 (Kerry Group Services Ltd.), HY-YEST441 (Kerry Group Services Ltd.), HY-YEST444 (Kerry Group Services Ltd.), HY-YEST455 (Kerry Group Services Ltd.), HY-YEST504 (Kerry Group Services Ltd.), or ULTRAPEP YE (Kerry Group Services Ltd.). In a further embodiment, the concentration of the yeast extract is between about 1 g / L and about 50 g / L, for example, between about 5 g / L and about 25 g / L, or about 10 g / L.

[0019] In certain embodiments, the medium comprises at least about 50 mM amino acids; a potassium salt, a carbon source, and optionally yeast extract.

[0020] In another embodiment, the medium comprises at least about 50 mM amino acids, between about 5.0 mM and about 15.0 mM glycine, between about 0.2 g / L and about 1.25 g / L of potassium salt, between about 25 g / L and about 100 g / L of a carbon source, and between about 5 g / L and about 25 g / L of yeast extract.

[0021] In a further embodiment, the medium comprises at least about 60 mM amino acids, about 7.5 mM glycine, about 0.9 g / L potassium chloride, 50 g / L glucose, and about 10 g / L ultrafiltered yeast extract.

[0022] A further aspect of the present invention relates to a method for growing polysaccharide-producing bacteria, comprising the steps of: a) adding a medium of the present invention to a bioreactor; b) inoculating the medium with polysaccharide-producing bacteria; and c) growing the bacteria by fermentation, wherein the growing comprises the constant addition of nutrients to the medium. In one embodiment, the nutrient is a carbon source, such as glucose. In one embodiment, growing is carried out until the bacteria exhibit a cell density, as determined by an optical density at 600 nm (OD), of at least 9.0. In another embodiment, the grown bacteria exhibit a cell density, as determined by an OD at 600 nm, of at least 9.0. In another embodiment, growing is carried out until the bacteria exhibit a polysaccharide concentration of at least about 250 mg / L. In another embodiment, the grown bacteria exhibit a polysaccharide concentration of at least about 250 mg / L. In a further embodiment, the polysaccharide-producing bacteria 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.

[0023] Yet another aspect of the present invention relates to a method for growing polysaccharide-producing bacteria, comprising: a) adding a medium according to the above description to a bioreactor; b) inoculating the medium with polysaccharide-producing bacteria; and c) growing the bacteria by perfusion, wherein growing comprises (i) removing spent medium from the culture, (ii) adding fresh medium, and (iii) maintaining the bacteria. In one embodiment, the perfusion rate is between about 0.07 and about 2.00 feed volumes to starting culture volume per hour (VVH), e.g., between about 0.67 and about 1.33 VVH, or about 1.20 VVH. In another embodiment, the perfusion rate is variable. For example, in one embodiment, perfusion begins at a first rate and is increased to a second rate. In another embodiment, perfusion begins at a first rate and is decreased to a second rate.

[0024] In one embodiment, the duration of perfusion is between about 1 hour and about 15 hours, for example, between about 1 hour and about 10 hours, or about 7 hours.

[0025] In another embodiment, cell growth of the grown bacteria is at least two-fold greater than cell growth in a batch fermentation system. In one embodiment, growth is carried out until the bacteria exhibit a cell density, as determined by an OD at 600 nm, of at least 20.0. In a further embodiment, the grown bacteria exhibit a cell density, as determined by an OD at 600 nm, of at least 20.0. In yet another embodiment, growth is carried out until the bacteria exhibit a polysaccharide concentration of at least about 600 mg / L. In yet another embodiment, the grown bacteria exhibit a polysaccharide concentration of at least about 600 mg / L.

[0026] In a further embodiment, the polysaccharide-producing bacteria 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. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention provides media and methods for the production of polysaccharides by bacterial cell culture. In particular, the present invention provides a system for maximizing the production of capsular polysaccharides of encapsulated bacteria.

[0028] Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0029] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference in their entirety.

[0030] Although the terms used herein have meanings that are recognized and known to those of ordinary skill in the art, for convenience and completeness, certain terms and their meanings are set forth below and throughout the specification.

[0031] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure and so forth.

[0032] The term "about" or "approximately" refers to a value within a statistically meaningful range. Such a range may be within one order of magnitude, typically within 20%, more typically even 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 understood by one of ordinary skill in the art. Whenever a range is referred to within this application, it is contemplated that any integer falling within that range is also an embodiment of the invention.

[0033] The term "batch culture," as used herein, refers to a method of culturing cells in which all components that will ultimately be used in culturing the cells, including the medium and the cells themselves, are provided at the beginning of the culture process. Batch cultures are typically stopped at some point, and the cells and / or components in the medium are harvested and optionally purified.

[0034] The term "bioreactor," as used herein, refers to any vessel used for the growth of bacterial cell cultures. Bioreactors can be of any size, so long as they are useful for culturing bacterial cells. Typically, bioreactors are at least 1 liter and can be 10; 50; 100; 250; 500; 1,000; 2,500; 5,000; 8,000; 10,000; or 12,000 liters or more, or any volume in between. Typically, the internal conditions of the bioreactor, including but not limited to pH and temperature, are controlled during the culture period. Bioreactors can be constructed of any material suitable for maintaining bacterial cell cultures suspended in a medium under the culture conditions of the present invention, including glass, plastic, or metal. The term "production bioreactor," as used herein, refers to the final bioreactor used in the production of a polysaccharide of interest. Large-scale cell culture production bioreactor volumes are typically at least 500 liters and can be 1,000; 2,500; 5,000; 8,000; 10,000; or 12,0000 liters or more, or any volume in between. One of skill in the art would know and be able to select an appropriate bioreactor for use in practicing the present invention.

[0035] The term "capsular polysaccharide" or "capsule polysaccharide" refers to a glycopolymer that contains repeating units of one or more monosaccharides joined by glycosidic linkages. Capsular polysaccharides typically form a capsule-like layer around the bacterial cell.

[0036] The term "cell density," as used herein, refers to the number of cells present in a given volume of medium.

[0037] 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 variables. This term, as used herein, also refers to the fraction of cells that are viable at a particular time relative to the total number of live and dead cells in the culture at that time.

[0038] The terms "comprises," "comprised," "comprising," "contains," "containing," and the like may have the meaning given to them in U.S. patent law, for example, they may mean "includes," "included," "including," etc. Such terms refer to the inclusion of a specified component or set of components without the exclusion of any other components.

[0039] The terms "consist of" and "consisting of" have the meaning ascribed to them in U.S. patent law, i.e., these terms are conditional. Thus, these terms refer to the inclusion of a specified component or set of components, and the exclusion of all other components.

[0040] Terms such as "consisting essentially of" and "consists essentially of" have the meaning given to them in U.S. patent law, e.g., they allow for the inclusion of additional components or steps that do not depart from the novel or basic characteristics of the invention; i.e., they exclude additional, unrecited components or steps that depart from the novel or basic characteristics of the invention, and they exclude components or steps of the prior art, e.g., documents in the art cited herein or incorporated by reference; among other reasons, because it is a goal of this description to define embodiments that are patentable, e.g., novel, unobvious inventions, over the prior art, e.g., documents cited herein or incorporated by reference.

[0041] The terms "culture," "cell culture," and "bacterial cell culture," as used herein, refer to a population of bacterial cells suspended in a medium under conditions suitable for the survival and / or growth of the cell population. These terms, as used herein, can refer to a combination comprising a bacterial cell population and the medium in which the population is suspended, as will be apparent to one of skill in the art.

[0042] The term "disaccharide," as used herein, refers to a polysaccharide composed of two monosaccharide units or moieties linked together by a glycosidic bond.

[0043] 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 following the initiation of the culturing process. The provided components typically include nutritional supplements for the cells that have been depleted during the culturing process. The fed-batch culture is typically stopped at some point, and the cells and / or components in the medium are harvested and optionally purified.

[0044] The terms "culture medium," "cell culture medium," "bacterial culture medium," and "culture medium," as used herein, refer to a solution containing nutrients that nourish growing bacterial cells. Typically, these solutions provide the essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by cells 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 preferably formulated to an optimal pH and salt concentration for cell survival and proliferation. The medium may also be a "defined medium," i.e., a serum-free medium that does not contain proteins, hydrolysates, or components of unknown composition. Defined media do not contain animal-derived components, and all components have known chemical structures.

[0045] The term "metabolic waste products," as used herein, refers to compounds produced by cell cultures as a result of normal or abnormal metabolic processes that are in some way harmful to the cell culture, particularly with respect to the production of capsular polysaccharides. For example, metabolic waste products may be harmful to the growth or viability of the cell culture or may reduce the amount of capsular polysaccharide produced. Exemplary metabolic waste products include lactate, which is produced as a result of glucose metabolism, and ammonium, which is produced as a result of glutamine metabolism. One goal of the present invention is the slowing down, reduction, or even elimination of metabolic waste product production in bacterial cell cultures.

[0046] "Monosaccharide," as used herein, refers to a single sugar residue in an oligosaccharide.

[0047] "Oligosaccharide," as used herein, refers to a compound containing two or more monosaccharide units or moieties. Within the context of an oligosaccharide, each monomer unit or moiety is a monosaccharide that is or can be linked to another monosaccharide unit or moiety through a hydroxyl group. Oligosaccharides can be prepared either by chemical synthesis from protected, single-residue sugars or by chemical degradation of biologically produced polysaccharides. Alternatively, oligosaccharides can be prepared by in vitro enzymatic methods.

[0048] The term "perfusion culture," as used herein, refers to a method of culturing cells in which additional components are continuously or semi-continuously provided to the culture following the initiation of the culturing process. The provided components typically include nutritional supplements for the cells that are depleted during the culturing process. A portion of the components, such as cells and / or metabolic waste products in the medium, are collected, typically in a continuous or semi-continuous manner, and optionally purified.

[0049] The term "polysaccharide" (PS) refers to a linear or branched polymer of at least five monosaccharide units or moieties. Specifically, larger numbers of repeating units are referred to herein as polysaccharides, where n is greater than about 5, e.g., greater than about 10.

[0050] The term "sugar," as used herein, refers to a single sugar moiety or monosaccharide unit, as well as combinations of two or more single sugar moieties or monosaccharide units covalently linked to form disaccharides, oligosaccharides, and polysaccharides. The term "sugar" can be used interchangeably with the term "carbohydrate."

[0051] The term "seeding," as used herein, refers to the process of providing a cell culture to a bioreactor or another vessel. The cells can be pre-grown in another bioreactor or vessel. Alternatively, the cells can be frozen and thawed just prior to providing the cells to the bioreactor or vessel. The term refers to any number of cells, including a single cell.

[0052] 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 milligrams of polysaccharide per liter of medium.

[0053] The terms "vaccine" or "vaccine composition" are used interchangeably and refer to a pharmaceutical composition containing at least one immunogenic composition that elicits an immune response in an animal.

[0054] bacteria Any bacterium having cell wall polysaccharides can be utilized in accordance with the present invention. In a preferred embodiment, the bacterium is an encapsulated bacterium. Non-limiting examples of encapsulated bacteria that can be used in accordance with the present invention 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 more preferred embodiment, the bacterium has fastidious growth requirements. Tricky bacteria include, but are not limited to, Streptococcus species (e.g., S. agalactiae and S. pneumoniae).

[0055] Streptococcus agalactiae (S. agalactiae), also known as group B streptococcus (GBS), has 10 different serotypes, all of which can be used in the present invention. These serotypes include Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX. All GBS capsular polysaccharides have a branched, repeating structure with terminal α2-3-linked sialic acid residues, which are necessary for bacterial pathogenicity.Some examples of GBS strains contemplated for use in the present invention include, but are not limited to, 090, A909 (ATCC Accession No. BAA-1138), 515 (ATCC Accession No. BAA-1177), B523, CJB524, MB4052 (ATCC Accession No. 31574), H36B (ATCC Accession No. 12401), S40, S42, MB4053 (ATCC Accession No. 31575), M709, 133, 7357, PFEGBST0267, MB4055 (ATCC accession number: 31576), 18RS21 (ATCC accession number: BAA-1175), S16, S20, V8 (ATCC accession number: 12973), DK21, DK23, UAB, 5401, PFEGBST0708, MB4082 (ATCC accession number: 31577), M132, 110, M781 (ATCC accession number: BAA-2 2), D136C(3) (ATCC accession number: 12403), M782, S23, 120, MB4316 (M-732; ATCC accession number: 31475), M132, K79, COH1 (ATCC accession number: BAA-1176), PFEGBST0563, 3139 (ATCC accession number: 49446), CZ-NI-016, PFEGBST0961, 1169-NT1, CJB111 (ATCC accession number: BAA-23), CJB112, 2603V / R (ATCC Accession No.: BAA-611), NCTC10 / 81, CJ11, PFEGBST0837, 118754, 114852, 114862, 114866, 118775, B4589, B4645, SS1214, CZ-PW-119, 7271, CZ-PW-045, JM9130013, JM9130672, IT-NI-016, IT-PW-62, and IT-PW-64.

[0056] There are over 90 different serotypes of S. pneumoniae, all of which are contemplated for use in the present invention. Examples include, but are not limited to, serotypes 1, 2, 3, 4, 5, 6A, 6B, 7A, 7C, 7F, 8, 9N, 9L, 9V, 10A, 10B, 11A, 11F, 12A, 12F, 14, 15A, 15B, 15C, 17A, 17F, 18C, 19A, 19F, 20, 22F, 23A, 23B, 23F, 24F, 33F, 35, 38, 39, 40, and 42. For example, in one embodiment, S. pneumoniae serotypes 8, 10A, 11A, 12F, 15B, 22F, or 33F can be used in the present invention. In another embodiment, serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F of S. pneumoniae can be used in the present invention.

[0057] Similarly, any encapsulated strain of S. aureus can be used in the present invention. Preferably, S. aureus strains that produce capsular polysaccharides of serotype 5 or 8 are contemplated, such as Reynolds, Becker, Newman, PS80, JL278, and JL812.

[0058] Any strain of N. meningitidis serogroups A, C, Y and W-135 can be used in the present invention.

[0059] Any strain of E. coli can be used in the present invention.

[0060] Any strain of Salmonella typhi (S. typhi) Vi can be used in the present invention.

[0061] Any strain of H. influenza type b can be used in the present invention.

[0062] Any strain of K. pneumoniae can be used in the present invention.

[0063] Enterococcus faecalis (E. faecalis) can be used in the present invention.

[0064] Exemplary strains of E. faecium that can be used in the present invention include those listed in Table 1.

[0065] [Table 1-1]

[0066] [Table 1-2]

[0067] Additionally, any number of commercially available and non-commercially available bacteria having cell wall polysaccharides can be utilized in accordance with the present invention. Those skilled in the art will recognize that some bacteria may have different nutritional requirements and / or require different culture conditions for optimal growth and will be able to modify the conditions as needed.

[0068] Often, strains of bacteria are selected or engineered to produce high levels of polysaccharides, hi some embodiments, bacterial cells are engineered to produce high levels of polysaccharides.

[0069] Cell culture medium The present invention provides a variety of media formulations that maximize polysaccharide production in bacterial cell cultures.

[0070] Complex media Bacterial cell cultures, particularly for fastidious bacteria and / or bacteria that produce cell wall polysaccharides, are often grown in complex media, such as Columbia broth, Luria-Bertani (LB) broth, Todd-Hewitt broth, GC medium, blood broth, or brain heart infusion broth. Therefore, complex media have been developed to maximize bacterial growth and polysaccharide production. In one aspect, the present invention relates to a complex culture medium comprising a plant hydrolysate, yeast extract, and a carbon source.

[0071] Suitable vegetable hydrolysates include, but are not limited to, HYPEP1510 (Kerry Group Services Ltd.), HYPEP4601 (Kerry Group Services Ltd.), HYPEP5603 (Kerry Group Services Ltd.), HY-SOY (Kerry Group Services Ltd.), AMI-SOY (Kerry Group Services Ltd.), NZ-SOY (Quest), NZ-SOY BL4 (Kerry Group Services Ltd.), NZ-SOY BL7 (Quest), SHEFTONE D (Kerry Group Services Ltd.), SE50M, SE50MK, soy peptone, BACTO soytone (Difco Laboratories Inc.), NUTRISOY2207 (ADM), NUTRISOY (ADM), NUTRISOY flour (ADM), and soybean meal. In a preferred embodiment, the vegetable hydrolysate is soybean hydrolysate. Preferably, the soy hydrolysate is HYPEP1510 (Kerry Group Services Ltd.).

[0072] The concentration of the plant hydrolysate in the culture medium is between about 5 g / L and about 75 g / L, for example, between about 5 g / L and about 65 g / L, between about 5 g / L and about 55 g / L, between about 5 g / L and about 45 g / L, between about 5 g / L and about 35 g / L, between about 10 g / L and about 70 g / L, between about 10 g / L and about 60 g / L, between about 10 g / L and about 50 g / L, The concentration of the plant hydrolysate in the culture medium can range from about 10 g / L to about 40 g / L, from about 15 g / L to about 75 g / L, from about 15 g / L to about 65 g / L, from about 15 g / L to about 55 g / L, from about 15 g / L to about 45 g / L, from about 20 g / L to about 70 g / L, from about 20 g / L to about 60 g / L, or from about 20 g / L to about 50 g / L. In a preferred embodiment, the concentration of the plant hydrolysate in the culture medium is between about 10 g / L and about 50 g / L, and most preferably about 28 g / L.

[0073] Suitable yeast extracts for use in the present invention include yeast autolysates, ultrafiltered yeast extracts, and synthetic yeast extracts. In one embodiment, the yeast extract is BD BBL (BD Biosciences), BD BACTO (BD Biosciences), HY YEST412 (Kerry Group Services Ltd.), HY YEST444 (Kerry Group Services Ltd.), HY-YEST441 (Kerry Group Services Ltd.), HY-YEST455 (Kerry Group Services Ltd.), or HY YEST504 (Kerry Group Services Ltd.). In another embodiment, the yeast extract is an ultrafiltered yeast extract, such as AMBERFERM5902 (Sensient Technologies Corp.), BD DIFCO (BD Biosciences), HYPEP YE (Kerry Group Services Ltd.), or ULTRAPEP YE (Kerry Group Services Ltd.). In a further embodiment, the yeast extract is a synthetic yeast extract, such as BD RECHARGE (BD Biosciences). Most preferably, the yeast extract is an ultrafiltered yeast extract, such as AMBERFERM5902 (Sensient Technologies Corp.).

[0074] The concentration of the yeast extract in the culture medium is about 1 g / L to about 50 g / L, for example, between about 1 g / L to about 40 g / L, between about 1 g / L to about 30 g / L, between about 1 g / L to about 25 g / L, between about 1 g / L to about 20 g / L, between about 1 g / L to about 15 g / L, between about 1 g / L to about 10 g / L, between about 5 g / L to about 50 g / L, between about 5 g / L to about 40 g / L, between about 5 g / L to about 30 g / L, between about 5 g / L to about 25 g / L, between about 5 g / L to about 20 g / L, The concentration of yeast extract in the culture medium can range from about 5 g / L to about 15 g / L, about 10 g / L to about 50 g / L, about 10 g / L to about 40 g / L, about 10 g / L to about 30 g / L, about 10 g / L to about 35 g / L, about 10 g / L to about 30 g / L, about 10 g / L to about 25 g / L, about 10 g / L to about 20 g / L, about 15 g / L to about 50 g / L, about 15 g / L to about 40 g / L, about 15 g / L to about 30 g / L, or about 15 g / L to about 25 g / L. In a preferred embodiment, the concentration of yeast extract in the culture medium is between about 5 g / L and about 25 g / L, and most preferably about 10 g / L.

[0075] Any carbon source can be used in the culture medium of the present invention. Suitable carbon sources include glucose, dextrose, mannitol, lactose, sucrose, fructose, galactose, raffinose, xylose, and / or mannose. Preferably, the carbon source in the culture medium is glucose.

[0076] The concentration of the carbon source in the culture medium can be in the range of about 25 g / L to about 100 g / L, for example, between about 25 g / L to about 90 g / L, between about 25 g / L to about 80 g / L, between about 25 g / L to about 70 g / L, between about 25 g / L to about 60 g / L, between about 25 g / L to about 50 g / L, between about 50 g / L to about 100 g / L, between about 50 g / L to about 90 g / L, between about 50 g / L to about 80 g / L, between about 50 g / L to about 70 g / L, between about 60 g / L to about 100 g / L, between about 60 g / L to about 90 g / L, between about 60 g / L to about 80 g / L, between about 70 g / L to about 100 g / L, or between about 70 g / L to about 90 g / L. In a preferred embodiment, the concentration of the carbon source in the culture medium is between about 50 g / L and about 90 g / L, and most preferably about 80 g / L.

[0077] Thus, the inventors have discovered that a combination of a plant hydrolysate, yeast extract, and a carbon source is useful for supporting maximum bacterial cell growth and polysaccharide production. In one aspect, the present invention relates to a culture medium comprising a plant hydrolysate, a yeast extract, and a carbon source. The plant hydrolysate can be any suitable plant hydrolysate known in the art, such as those described above. Preferably, the hydrolysate is a soy hydrolysate. More preferably, the soy hydrolysate is HYPEP1510 (Kerry Group Services Ltd.). Any yeast extract known in the art, such as those described above, can be used. In a preferred embodiment, the yeast extract is AMBERFERM5902 (Sensient Technologies Corp.).

[0078] In one embodiment, a complex culture medium of the present invention can include a phosphate-containing component, such as Na2HPO4, K2HPO4, or KH2PO4.

[0079] In alternative embodiments, the culture medium can contain a variety of other factors known in the art to enhance growth, such as amino acids, vitamins, nucleosides, and inorganic salts.

[0080] In yet another aspect, growth is carried out using any of the methods disclosed herein until a bacterial cell culture using a complex medium of the invention has a cell density, as determined by optical density at 600 nm (OD), of at least 15.0, e.g., at least 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, or 30.0. In a preferred embodiment, growth is carried out until the cell density is at least 15.0. In yet another aspect, bacterial cell cultures using the complex media of the present invention can have a cell density, as determined by optical density at 600 nm (OD), of at least 15.0, e.g., at least 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, or 30.0. In a preferred embodiment, the cell density is at least 15.0.

[0081] The yield of GBS polysaccharides can be determined by measuring the concentration of sialic acid. Sialic acid is released from cell-bound polysaccharides by digesting pelleted cells using methods known in the art. The digest can be assayed by anion exchange chromatography (AEX) using high-performance liquid chromatography (HPLC). The polysaccharide concentration is then determined by multiplying the sialic acid value by a repeating unit weight conversion factor. For example, the conversion factors for each GBS serotype are as follows: Ia, Ib, and III = 3.24; II and V = 4.29; and IV = 3.77. Quantification of polysaccharides for S. pneumoniae or other encapsulated bacteria is performed by first releasing the capsular polysaccharides from the cell wall by treatment with detergents, such as sodium deoxycholate (DOC) or sodium N-laurylsarcosinate (NLS); acid treatment at high temperature; base treatment; and / or mechanical lysis. The released polysaccharides in the crude lysate are then assayed against certified standards using size exclusion chromatography (SEC) HPLC.

[0082] In one aspect, using any of the methods disclosed herein, bacterial cell cultures using complex media of the invention are grown until they reach a polysaccharide concentration, as determined by the concentration of sialic acid, of at least about 200 mg / L, e.g., at least about 250 mg / L; 300 mg / L; 350 mg / L; 400 mg / L; 450 mg / L; 500 mg / L; 550 mg / L; 600 mg / L; 650 mg / L; or 700 mg / L. In a preferred embodiment, growth is carried out until the polysaccharide concentration is at least about 600 mg / L. In one aspect, bacterial cell cultures using the complex media of the present invention can have a polysaccharide concentration, as determined by the concentration of sialic acid, of at least about 200 mg / L, e.g., at least about 250 mg / L; 300 mg / L; 350 mg / L; 400 mg / L; 450 mg / L; 500 mg / L; 550 mg / L; 600 mg / L; 650 mg / L; or 700 mg / L. In a preferred embodiment, the polysaccharide concentration is at least about 600 mg / L.

[0083] Defined medium However, given the potential for inconsistency in complex media, chemically defined media were also explored to maximize bacterial growth and polysaccharide production. Surprisingly, applicant's proprietary media for culturing mammalian cells were discovered to unexpectedly enhance both cell growth and polysaccharide production; these media are disclosed in U.S. Pat. No. 7,294,484, the entire contents of which are incorporated herein by reference. Specifically, the inventors discovered that defined media having a total amino acid concentration greater than about 50 mM unexpectedly enhanced both cell growth and polysaccharide production. Exemplary media for culturing mammalian cells are shown in Table 2 below. Compared to the media formulations of the present invention, conventional media formulations start with relatively low levels of total amino acids. For example, a conventional medium for culturing cells known as DME-F12 (a 50:50 mixture of Dulbecco's Modified Eagle's Medium and Ham's F12 medium) has a total amino acid content of 7.29 mM, and a conventional medium for culturing cells known as RPMI-1640 has a total amino acid content of 6.44 mM (see, e.g., H. J. Morton, In Vitro, 6:89-108 (1970); R. G. Ham, Proc. Nat. Assoc. Sci. (USA), 53:288-293 (1965); G. E. Moore et al., J. Am. Medical Assn., 199:519-24 (1967), all of which are incorporated herein by reference).

[0084] [Table 2-1]

[0085] [Table 2-2]

[0086] Therefore, the present invention relates to a medium for culturing cells having a total amino acid concentration of at least about 50 mM, for example, at least about 55 mM, at least 60 mM, at least 70 mM, and at least 75 mM. In a preferred embodiment, the total amino acid concentration is at least 60 mM.

[0087] In one embodiment of the present invention, the total concentration of glycine in the medium for culturing bacterial cells is between about 1.5 mM and about 60.0 mM, for example, between about 1.5 mM and about 50.0 mM, between about 1.5 mM and about 40.0 mM, between about 1.5 mM and about 30.0 mM, between about 1.5 mM and about 20.0 mM, between about 1.5 mM and about 15.0 mM, between about 1.5 mM and about 10.0 mM, between about 1.5 mM and about 7.5 mM, between about 1.5 mM and about 5.0 mM, between about 5.0 mM and about 60.0 mM, between about 5.0 mM and about 50.0 mM, or about 5. The concentration may range from about 0 mM to about 40.0 mM, from about 5.0 mM to about 30.0 mM, from about 5.0 mM to about 20.0 mM, from about 5.0 mM to about 15.0 mM, from about 5.0 mM to about 10.0 mM, from about 5.0 mM to about 7.5 mM, from about 7.5 mM to about 60.0 mM, from about 7.5 mM to about 50.0 mM, from about 7.5 mM to about 40.0 mM, from about 7.5 mM to about 30.0 mM, from about 7.5 mM to about 20.0 mM, from about 7.5 mM to about 15.0 mM, or from about 7.5 mM to about 10.0 mM. In a preferred embodiment, the total concentration of glycine in the medium in which the bacterial cells are cultured is between about 5.0 mM and about 15.0 mM, and most preferably about 7.5 mM.

[0088] In one embodiment of the present invention, the total concentration of arginine in the medium for culturing bacterial cells is between about 1.0 mM and about 30.0 mM, for example, between about 1.0 mM and about 20.0 mM, between about 1.0 mM and about 15.0 mM, between about 1.0 mM and about 10.0 mM, between about 1.0 mM and about 7.5 mM, between about 1.0 mM and about 5.0 mM, between about 4.0 mM and about 20.0 mM, or between about 4.0 mM and about 15.0 mM. The total arginine concentration in the medium for culturing bacterial cells can range from about 1.0 mM to about 20.0 mM, about 4.0 mM to about 10.0 mM, about 4.0 mM to about 7.5 mM, about 10.0 mM to about 30.0 mM, about 10.0 mM to about 25.0 mM, about 10.0 mM to about 20.0 mM, about 10.0 mM to about 15.0 mM, about 15.0 mM to about 30.0 mM, about 15.0 mM to about 25.0 mM, or about 15.0 mM to about 20.0 mM. In a preferred embodiment, the total arginine concentration in the medium for culturing bacterial cells is between about 1.0 mM and about 20.0 mM, and most preferably about 4.0 mM.

[0089] In one embodiment of the present invention, the total concentration of cysteine ​​in the medium for culturing bacterial cells can be between about 0.1 mM and about 5.0 mM, for example, between about 0.1 mM and about 4.5 mM, between about 0.1 mM and about 4.0 mM, between about 0.1 mM and about 3.5 mM, between about 0.1 mM and about 3.0 mM, between about 0.1 mM and about 2.5 mM, between about 0.4 mM and about 5.0 mM, between about 0.4 mM and about 4.5 mM, between about 0.4 mM and about 4.0 mM, between about 0.4 mM and about 3.5 mM, between about 0.4 mM and about 3.0 mM, between about 0.4 mM and about 2.5 mM, or between about 0.4 mM and about 2.0 mM. In a preferred embodiment, the total concentration of cysteine ​​in the medium in which the bacterial cells are cultured is between about 0.1 mM and about 3.5 mM, and most preferably about 0.4 mM.

[0090] In one embodiment of the present invention, the total concentration of serine in the medium for culturing bacterial cells is between about 5.0 mM and about 75.0 mM, for example, between about 5.0 mM and about 50.0 mM, between about 5.0 mM and about 40.0 mM, between about 5.0 mM and about 30.0 mM, between about 5.0 mM and about 20.0 mM, between about 5.0 mM and about 20.0 mM, between about 5.0 mM and about 15.0 mM, or between about 10.0 mM and about 75.0 mM. The total concentration of serine in the medium for culturing bacterial cells can be between about 5.0 mM and about 15.0 mM, and most preferably about 7.5 mM.

[0091] In one embodiment of the present invention, the total concentration of glutamine in the medium for culturing bacterial cells is between about 1.0 mM and about 30.0 mM, for example, between about 1.0 mM and about 20.0 mM, between about 1.0 mM and about 15.0 mM, between about 1.0 mM and about 10.0 mM, between about 1.0 mM and about 7.5 mM, between about 1.0 mM and about 5.0 mM, between about 4.0 mM and about 20.0 mM, or between about 4.0 mM and about 15.0 mM. The total concentration of glutamine in the medium for culturing bacterial cells can range from about 1.0 mM to about 20.0 mM, about 4.0 mM to about 10.0 mM, about 4.0 mM to about 7.5 mM, about 10.0 mM to about 30.0 mM, about 10.0 mM to about 25.0 mM, about 10.0 mM to about 20.0 mM, about 10.0 mM to about 15.0 mM, about 15.0 mM to about 30.0 mM, about 15.0 mM to about 25.0 mM, or about 15.0 mM to about 20.0 mM. In a preferred embodiment, the total concentration of glutamine in the medium for culturing bacterial cells is between about 1.0 mM and about 20.0 mM, and most preferably about 4.0 mM.

[0092] In one embodiment of the present invention, the total concentration of tyrosine in the medium for culturing bacterial cells can be in the range of about 0.1 mM to about 5.0 mM, for example, about 0.1 mM to about 4.5 mM, about 0.1 mM to about 4.0 mM, about 0.1 mM to about 3.5 mM, about 0.1 mM to about 3.0 mM, about 0.1 mM to about 2.5 mM, about 1.0 mM to about 5.0 mM, about 1.0 mM to about 4.5 mM, about 1.0 mM to about 4.0 mM, about 1.0 mM to about 3.5 mM, about 1.0 mM to about 3.0 mM, about 1.0 mM to about 2.5 mM, or about 1.0 mM to about 2.0 mM. In a preferred embodiment, the total concentration of tyrosine in the medium in which the bacterial cells are cultured is between about 1.0 mM and about 3.5 mM, and most preferably about 2.9 mM or about 3.0 mM.

[0093] In one embodiment of the present invention, the total concentration of asparagine in a medium for culturing bacterial cells is between about 5.0 mM and about 50.0 mM, for example, between about 5.0 mM and about 40.0 mM, between about 5.0 mM and about 30.0 mM, between about 5.0 mM and about 25.0 mM, between about 5.0 mM and about 20.0 mM, between about 5.0 mM and about 15.0 mM, between about 5.0 mM and about 10.0 mM, or between about 10.0 mM and about 50.0 mM. The total concentration of asparagine in the medium for culturing bacterial cells can be between about 10.0 mM and about 40.0 mM, between about 10.0 mM and about 30.0 mM, between about 10.0 mM and about 25.0 mM, between about 10.0 mM and about 20.0 mM, between about 15.0 mM and about 50.0 mM, between about 15.0 mM and about 40.0 mM, between about 15.0 mM and about 30.0 mM, between about 15.0 mM and about 25.0 mM, or between about 15.0 mM and about 20.0 mM. In a preferred embodiment, the total concentration of asparagine in the medium for culturing bacterial cells is between about 10.0 mM and about 30.0 mM, and most preferably about 20.0 mM.

[0094] In another aspect of the invention, the medium in which the cells are cultured does not contain asparagine.

[0095] The inventors have also found that potassium is a salt beneficial to polysaccharide production and is unrelated to growth. Thus, in one aspect of the invention, the medium in which the cells are cultured contains a potassium salt, such as potassium chloride or potassium sulfate.

[0096] In one embodiment, the concentration of potassium salt in the medium for culturing cells is between about 0.1 g / L and about 25 g / L, for example, between about 0.1 g / L and about 20 g / L, between about 0.1 g / L and about 10 g / L, between about 0.1 g / L and about 5 g / L, between about 0.1 g / L and about 1.5 g / L, between about 0.1 g / L and about 1.25 g / L, or between about 0.1 g / L and about 1. Between 0g / L, between about 0.1g / L and about 0.9g / L, between about 0.1g / L and about 0.8g / L, between about 0.1g / L and about 0.7g / L, between about 0.1g / L and about 0.6g / L, between about 0.1g / L and about 0.5g / L, between about 0.2g / L and about 1.5g / L, between about 0.2g / L and about 1.25g / L, between about 0.2g / L and about 1.0g / L Between about 0.2g / L and about 0.9g / L, Between about 0.2g / L and about 0.8g / L, Between about 0.2g / L and about 0.7g / L, Between about 0.2g / L and about 0.6g / L, Between about 0.2g / L and about 0.5g / L, Between about 0.3g / L and about 1.5g / L, Between about 0.3g / L and about 1.25g / L, Between about 0.3g / L and about 1.0g / L, Between about 0. The total concentration of potassium salts in the medium for culturing bacterial cells can be between about 3 g / L and about 0.9 g / L, between about 0.3 g / L and about 0.8 g / L, between about 0.3 g / L and about 0.7 g / L, between about 0.3 g / L and about 0.6 g / L, between about 0.3 g / L and about 0.5 g / L, between about 0.5 g / L and about 1.5 g / L, between about 0.5 g / L and about 1.25 g / L, or between about 0.5 g / L and about 1.0 g / L. In a preferred embodiment, the total concentration of potassium salts in the medium for culturing bacterial cells is between about 0.2 g / L and about 1.25 g / L, and most preferably about 0.9 g / L.

[0097] In one aspect of the present invention, the medium for culturing cells of the present invention contains a carbon source. Suitable carbon sources include glucose, dextrose, mannitol, lactose, sucrose, fructose, galactose, raffinose, xylose, and / or mannose. In a preferred embodiment, the carbon source is glucose.

[0098] The total concentration of the carbon source in the medium for culturing bacterial cells is between about 25 g / L and about 100 g / L, for example, between about 25 g / L and about 90 g / L, between about 25 g / L and about 80 g / L, between about 25 g / L and about 70 g / L, between about 25 g / L and about 60 g / L, between about 25 g / L and about 50 g / L, or between about 50 g / L and about 100 g / L. The carbon source concentration can range from about 50 g / L to about 90 g / L, from about 50 g / L to about 80 g / L, from about 50 g / L to about 70 g / L, from about 60 g / L to about 100 g / L, from about 60 g / L to about 90 g / L, from about 60 g / L to about 80 g / L, from about 70 g / L to about 100 g / L, or from about 70 g / L to about 90 g / L. In a preferred embodiment, the concentration of the carbon source in the culture medium is between about 25 g / L to about 80 g / L, and most preferably about 50 g / L.

[0099] In another aspect, the medium in which cells are cultured is modified to match the sodium bicarbonate requirement of the anaerobically grown bacteria. Some examples of anaerobically grown polysaccharide-producing bacteria include Streptococcus agalactiae (S. agalactiae) and Streptococcus pneumoniae (S. pneumoniae). In one embodiment, about 0.1 g / L to about 20 g / L of sodium bicarbonate is added to the medium. For example, the sodium bicarbonate concentration may be between about 0.1 g / L and about 15 g / L, between about 0.1 g / L and about 10 g / L, between about 0.1 g / L and about 5.0 g / L, between about 0.1 g / L and about 3.0 g / L, between about 0.1 g / L and about 2.0 g / L, between about 0.1 g / L and about 1.25 g / L, between about 0.1 g / L and about 1.0 g / L, or between about 0.1 g / L and about 2.0 g / L. Between about 0.9g / L, between about 0.1g / L and about 0.8g / L, between about 0.1g / L and about 0.7g / L, between about 0.1g / L and about 0.6g / L, between about 0.1g / L and about 0.5g / L, between about 0.5g / L and about 20g / L, between about 0.5g / L and about 15g / L, between about 0.5g / L and about 10g / L, between about 0.5g / L and about 5.0g / L, Between 0.5g / L and approximately 3.0g / L, between approximately 0.5g / L and approximately 2.0g / L, between approximately 0.5g / L and approximately 1.25g / L, between approximately 0.5g / L and approximately 1.0g / L, between approximately 0.5g / L and approximately 0.9g / L, between approximately 0.5g / L and approximately 0.8g / L, between approximately 0.5g / L and approximately 0.7g / L, between approximately 0.75g / L and approximately 20g / L, between approximately 0.75g / L and approximately 20g / L The sodium bicarbonate concentration can be between about 0.75 g / L and about 15 g / L, between about 0.75 g / L and about 10 g / L, between about 0.75 g / L and about 5.0 g / L, between about 0.75 g / L and about 3.0 g / L, between about 0.75 g / L and about 2.0 g / L, between about 0.75 g / L and about 1.25 g / L, between about 0.75 g / L and about 1.0 g / L, or between about 0.75 g / L and about 0.9 g / L. Preferably, the sodium bicarbonate concentration is about 0.84 g / L, or between about 1.8 g / L and about 2.4 g / L.

[0100] In one embodiment of the present invention, the defined medium for culturing bacterial cells contains yeast extract. Yeast extracts suitable for use in the present invention can include yeast autolysate, ultrafiltered yeast extract, and synthetic yeast extract. In one embodiment, the yeast extract is BD BBL (BD Biosciences), BD BACTO (BD Biosciences), HY YEST412 (Kerry Group Services Ltd.), HY YEST441 (Kerry, Inc., Kerry Group Services Ltd.), HY YEST444 (Kerry Group Services Ltd.), or HY YEST504 (Kerry Group Services Ltd.). In another embodiment, the yeast extract is an ultrafiltered yeast extract, such as AMBERFERM5902 (Sensient Technologies Corp.), BD DIFCO (BD Biosciences), HYPEP YE (Kerry Group Services Ltd.), or ULTRAPEP YE (Kerry Group Services Ltd.). In a further embodiment, the yeast extract is a synthetic yeast extract, such as BD RECHARGE (BD Biosciences). Most preferably, the yeast extract is an ultrafiltered yeast extract, such as AMBERFERM5902 (Sensient Technologies Corp.).

[0101] The concentration of the yeast extract in the culture medium is between about 1 g / L and about 50 g / L, for example, between about 1 g / L and about 40 g / L, between about 1 g / L and about 30 g / L, between about 1 g / L and about 25 g / L, between about 1 g / L and about 20 g / L, between about 1 g / L and about 15 g / L, between about 1 g / L and about 10 g / L, between about 5 g / L and about 50 g / L, between about 5 g / L and about 40 g / L, between about 5 g / L and about 30 g / L, between about 5 g / L and about 25 g / L, or between about 5 g / L and about 20 g / L. The concentration of yeast extract in the culture medium may be between about 5 g / L and about 15 g / L, between about 10 g / L and about 50 g / L, between about 10 g / L and about 40 g / L, between about 10 g / L and about 30 g / L, between about 10 g / L and about 35 g / L, between about 10 g / L and about 30 g / L, between about 10 g / L and about 25 g / L, between about 10 g / L and about 20 g / L, between about 15 g / L and about 50 g / L, between about 15 g / L and about 40 g / L, between about 15 g / L and about 30 g / L, or between about 15 g / L and about 25 g / L. In a preferred embodiment, the concentration of yeast extract in the culture medium is between about 5 g / L and about 25 g / L, and most preferably about 10 g / L.

[0102] One aspect of the invention relates to a defined medium for culturing cells, comprising at least about 50 mM of an amino acid; a potassium salt, a carbon source, and optionally, yeast extract.

[0103] In one embodiment, the medium in which the cells are cultured comprises at least about 50 mM amino acids, between about 5.0 mM and about 15.0 mM glycine, between about 0.2 g / L and about 1.25 g / L of a potassium salt, between about 25 g / L and about 80 g / L of a carbon source, and between about 5 g / L and about 25 g / L of yeast extract.

[0104] In a preferred embodiment, the medium in which the cells are cultured comprises at least about 60 mM amino acids, about 7.5 mM glycine, about 0.9 g / L potassium chloride, 50 g / L glucose, and about 10 g / L ultrafiltered yeast extract.

[0105] Furthermore, one skilled in the art will recognize that any of the above listed conditions can be used either alone or in various combinations with one another. By utilizing a media formulation exhibiting one, some, or all of the above characteristics, one skilled in the art will be able to optimize cell growth and / or viability, as well as maximize polysaccharide production.

[0106] Any of these media formulations disclosed in this invention can be optionally supplemented with specific ions (e.g., sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, trace elements (inorganic compounds that are usually present at very low final concentrations), amino acids, lipids, protein hydrolysates, or glucose or other energy sources, as needed. These optional supplements can be added at the beginning of the culture or can be added at a later time to replenish depleted nutrients or for other reasons. Any desirable or necessary supplements that can be included in the disclosed media formulations will be understood by those skilled in the art.

[0107] In another aspect, growth is carried out by any of the methods disclosed herein until a bacterial cell culture using a defined medium of the invention has a cell density, as determined by optical density at 600 nm (OD), of at least 9.0, e.g., at least 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, or 20.0. In a preferred embodiment, growth is carried out by any of the methods disclosed herein until a cell density of at least 9.0 is achieved.

[0108] For sialic acid-containing polysaccharides, such as GBS, the polysaccharide yield can be determined by measuring the concentration of sialic acid. The pelleted cells are digested by methods known in the art to release sialic acid from the cell-bound polysaccharides. The digest is assayed by anion exchange chromatography (AEX) using high-performance liquid chromatography (HPLC). The polysaccharide concentration is then determined by multiplying the sialic acid value by a repeating unit weight conversion factor. For example, the conversion factors for each serotype of GBS are as follows: Ia, Ib, and III = 3.24; II and V = 4.29; and IV = 3.77.

[0109] In one aspect, growth is carried out using any of the methods disclosed herein until a bacterial cell culture using a defined medium of the invention has a polysaccharide concentration, as determined by the concentration of sialic acid, of at least about 250 mg / L, e.g., at least about 300 mg / L, 350 mg / L, 400 mg / L, 450 mg / L, 500 mg / L, 550 mg / L, 600 mg / L, 650 mg / L, 700 mg / L, 750 mg / L, 800 mg / L, 900 mg / L, 1000 mg / L, 1200 mg / L, 1500 mg / L, or 2000 mg / L. In a preferred embodiment, growth is carried out using any of the methods disclosed herein until the polysaccharide concentration is at least about 250 mg / L. In one aspect, bacterial cell cultures using defined media of the invention can have a polysaccharide concentration, as determined by the concentration of sialic acid, of at least about 250 mg / L, e.g., at least about 300 mg / L, 350 mg / L, 400 mg / L, 450 mg / L, 500 mg / L, 550 mg / L, 600 mg / L, 650 mg / L, 700 mg / L, 750 mg / L, 800 mg / L, 900 mg / L, 1000 mg / L, 1200 mg / L, 1500 mg / L, or 2000 mg / L. In a preferred embodiment, the polysaccharide concentration is at least about 250 mg / L.

[0110] Fermentation method The present invention provides fermentation methods for growing bacteria that produce polysaccharides. In one aspect, the growth methods of the present invention are used in combination with complex and defined media as described herein to maximize polysaccharide production.

[0111] Seed growth In one embodiment, growth of the polysaccharide-producing bacteria in the method of the invention proceeds in at least two phases: a seed growth phase and a fermentation phase. A seed culture is first grown using an inoculum from a stock culture, e.g., a working cell bank. This seed is used to inoculate either a second seed culture or a larger fermentation culture. As understood in the art, the number of seed cultures used can depend, for example, on the size and volume of the fermentation step.

[0112] Thus, in one aspect, the present invention relates to a method for culturing polysaccharide-producing bacteria. The method comprises culturing polysaccharide-producing bacterial cells in a first culture medium under conditions that promote cell growth; after the first culturing step, inoculating a second culture medium with all or a portion of the first medium; and culturing the inoculated second medium under conditions that promote cell growth and / or polysaccharide production. The method can further comprise isolating polysaccharides from the 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 comprises a culture medium according to the above description and an inoculation from a stock culture grown in the medium. In one embodiment, the first and second culture media are the same. In another embodiment, the first and second culture media are different.

[0113] Generally, one or more phases of seed growth are performed to scale up the amount of microorganisms derived from a stored culture so that they can be used as inoculum for the fermentation phase. The volume and amount of viable cells used to inoculate a fermentation culture can be precisely controlled when harvested from an actively growing culture (e.g., a seed culture) rather than from a stored culture.

[0114] Additionally, more than one (e.g., two or three) phases of seed growth can be used to inoculate the fermentation medium to scale up the amount of polysaccharide-producing bacteria. Alternatively, if desired, growth of the polysaccharide-producing bacteria can be directly initiated during the fermentation phase by inoculating directly from a stored culture.

[0115] To initiate the fermentation phase, some or all of the seed culture containing the polysaccharide-producing bacteria can be used to inoculate the fermentation culture medium. The appropriate concentration of seed culture to use to inoculate the fermentation medium can be determined by one of ordinary skill in the art.

[0116] In large-scale environments, fermentation can be used to maximize cell growth and / or polysaccharide production. In one embodiment, the polysaccharide-producing bacteria are grown as a fermentation culture. In one embodiment, the fermentation culture is inoculated from a seed culture grown in a first medium, and the fermentation culture is carried out in a second medium. In one embodiment, the second medium can be a complex or defined medium as described above. In another embodiment, the first and second medium are the same.

[0117] Fed-batch fermentation process In one embodiment, the polysaccharide-producing bacterial cells are cultured in a fed-batch system using the complex and defined media described above. In a fed-batch system, the culture is initiated by inoculating the cells, supplemented with the addition of at least one nutrient during the culture, and terminated with a single harvest of the cells. In one embodiment, the nutrients are added at a constant rate.

[0118] In one aspect, the carbon source is a nutrient added during cultivation. The carbon source can be any of the carbon sources described above for complex and / or defined media. In a preferred embodiment, the carbon source is glucose.

[0119] In some embodiments of the present invention, the amount of carbon source to be batched / the amount of carbon source to be fed can be about 10% / 90%, 15% / 85%, 20% / 80%, 25% / 75%, or 30% / 70%. For example, in a preferred embodiment, 20% of the total concentration of the carbon source is batched, and the remaining 80% is fed at a constant rate over the course of the culture. In another embodiment, 20% of the total concentration of the carbon source can be batched, and the remaining carbon source can be fed at a non-constant rate over the course of the culture.

[0120] In yet another aspect, the process of fed-batch fermentation is carried out until the bacterial cell culture has a cell density, as determined by optical density at 600 nm (OD), of at least 9.0, e.g., at least 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, or 20.0. In a preferred embodiment, the process of fed-batch fermentation is carried out until the cell density is at least 9.0.

[0121] In yet another aspect, the cell density of the bacterial cell culture in the fed-batch system of the invention, as determined by optical density at 600 nm (OD), can be at least 9.0, e.g., at least 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, or 20.0. In a preferred embodiment, the cell density is at least 9.0.

[0122] For sialic acid-containing polysaccharides, such as GBS, the polysaccharide yield can be determined by measuring the concentration of sialic acid. Pelleted cells are digested by methods known in the art to release sialic acid from the cell-bound polysaccharides. The digest is assayed by anion exchange chromatography (AEX) using high-performance liquid chromatography (HPLC). The polysaccharide concentration is then determined by multiplying the sialic acid value by a repeating unit weight conversion factor. For example, the conversion factors for each serotype of GBS are as follows: Ia, Ib, and III = 3.24; II and V = 4.29; and IV = 3.77. Polysaccharide yield for S. pneumoniae or other encapsulated bacteria can be quantified by first releasing the capsular polysaccharide from the cell wall by treatment with detergents, such as sodium deoxycholate (DOC) or sodium N-laurylsarcosinate (NLS); acid treatment at high temperature; base treatment; and / or mechanical lysis. The released polysaccharides in the crude lysate are then assayed against certified standards using size exclusion chromatography (SEC) HPLC.

[0123] In one aspect, the fed-batch fermentation process is carried out until the bacterial cell culture has a polysaccharide concentration, as determined by the concentration of sialic acid, of at least about 250 mg / L, e.g., at least about 300 mg / L, 350 mg / L, 400 mg / L, 450 mg / L, 500 mg / L, 550 mg / L, 600 mg / L, 650 mg / L, 700 mg / L, 750 mg / L, 800 mg / L, 900 mg / L, 1000 mg / L, 1200 mg / L, 1500 mg / L, or 2000 mg / L. In a preferred embodiment, the fed-batch fermentation process is carried out until the polysaccharide concentration is at least about 250 mg / L.

[0124] In one aspect, bacterial cell cultures produced by the fed-batch systems of the invention can have a polysaccharide concentration, as determined by SEC HPLC, of ​​at least about 250 mg / L, e.g., at least about 300 mg / L, 350 mg / L, 400 mg / L, 450 mg / L, 500 mg / L, 550 mg / L, 600 mg / L, 650 mg / L, 700 mg / L, 750 mg / L, 800 mg / L, 900 mg / L, 1000 mg / L, 1200 mg / L, 1500 mg / L, or 2000 mg / L. In a preferred embodiment, the polysaccharide concentration is at least about 250 mg / L.

[0125] The perfusion fermentation process In one embodiment, polysaccharide-producing bacterial cells are cultured in a perfusion culture system. The inventors have discovered that maximum polysaccharide production can be achieved in perfusion culture using the complex and defined media described above. An advantage of the perfusion system is that fresh medium can be added continuously. Furthermore, this system also allows metabolic waste products to be removed during production while maintaining cell viability.

[0126] Perfusion culture systems can include providing fresh medium to the cells and simultaneously removing spent medium; the spent medium is substantially free of cells or contains a concentration of cells substantially lower than the concentration of cells in the bioreactor. In perfusion culture, cells can be retained, for example, by filtration, ultrasonic filtration, centrifugation, or sedimentation.

[0127] In one embodiment, the spent medium is separated from the cells and removed, while the cells are retained in the bioreactor or returned to the bioreactor. The separation step can be normal flow filtration and / or cross-flow filtration. In one embodiment, the filtration system comprises a hollow fiber filter. In another embodiment, the 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 by a sedimentation system.

[0128] In one embodiment, the perfusion rate is between about 0.07 VVH and about 2.00 VVH, for example, between about 0.07 VVH and about 1.33 VVH, between about 0.07 VVH and about 1.20 VVH, between about 0.07 VVH and about 1.07 VVH, between about 0.07 VVH and about 0.93 VVH, between about 0.07 VVH and about 0.80 VVH, between about 0.07 VVH and about 0.67 VVH, between about 0.07 VVH and about 0.53 VVH, between about 0.07 VVH and about 0.40 VVH, between about 0.07 VVH and about 0.27 VVH, between about 0.13 VVH and about 2.0 ... Between about 1.33VVH, between about 0.13VVH and about 1.20VVH, between about 0.13VVH and about 1.07VVH, between about 0.13VVH and about 0.93VVH, between about 0.13VVH and about 0.80VVH, between about 0.13VVH and about 0.67VVH, between about 0.13VVH and about 0.53VVH, between about 0.13VVH and about 0.40VVH, between about 0.13VVH and about 0.27VVH, between about 0.27VVH and about 2.00VVH, between about 0.27VVH and about 1.33VVH, between about 0.27VVH and about 1.20VVH, between about 0.27VVH and about 1.07VVH between about 0.27VVH and about 0.93VVH, between about 0.27VVH and about 0.80VVH, between about 0.27VVH and about 0.67VVH, between about 0.27VVH and about 0.53VVH, between about 0.27VVH and about 0.40VVH, between about 0.40VVH and about 2.00VVH, between about 0.40VVH and about 1.33VVH, between about 0.40VVH and about 1.20VVH, between about 0.40VVH and about 1.07VVH, between about 0.40VVH and about 0.93VVH, between about 0.40VVH and about 0.80VVH, between about 0.40VVH and about 0.67VVH, between about 0.53V Between VH and approximately 2.00VVH, between approximately 0.53VVH and approximately 1.33VVH, between approximately 0.53VVH and approximately 1.20VVH, between approximately 0.53VVH and approximately 1.07VVH, between approximately 0.53VVH and approximately 0.93VVH, between approximately 0.53VVH and approximately 0.80VVH, between approximately 0.53VVH and approximately 0.67VVH, between approximately 0.67VVH and approximately 2.00VVH, between approximately 0.67VVH and approximately 1.33VVH, between approximately 0.67VVH and approximately 1.20VVH, between approximately 0.67VVH and approximately 1.07VVH, between approximately 0.67VVH and approximately 0.93VVH, or between approximately 0.67VVH and approximately 0.The perfusion rate may be between about 0.67 VVH and about 1.33 VVH, preferably about 1.20 VVH.

[0129] In one aspect, the duration of perfusion culture can be about 1 hour to about 15 hours, e.g., about 1 hour to about 14 hours, about 1 hour to about 13 hours, about 1 hour to about 12 hours, about 1 hour to about 11 hours, about 1 hour to about 10 hours, about 1 hour to about 9 hours, about 1 hour to about 8 hours, about 1 hour to about 7 hours, about 1 hour to about 6 hours, about 1 hour to about 5 hours, about 5 hours to about 15 hours, about 5 hours to about 14 hours, about 5 hours to about 13 hours, about 5 hours to about 12 hours, about 5 hours to about 11 hours, about 5 hours to about 10 hours, about 5 hours to about 9 hours, about 5 hours to about 8 hours, or about 5 hours to about 7 hours. In one embodiment, the duration of perfusion culture is about 1 hour to about 10 hours, preferably about 7 hours.

[0130] In one particular aspect, the perfusion rate can be variable (increased or decreased) over the duration of the culture. In one embodiment, the perfusion system starts at a first rate and increases the rate to a second rate. In another embodiment, the perfusion system starts at a first rate and decreases the rate to a second rate. In additional embodiments, the perfusion rate can be changed multiple times.

[0131] In one aspect, the perfusion rate is kept constant for the duration of the culture.

[0132] In another aspect of the invention, cell growth in a perfusion system can be at least 1.1 times, e.g., 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 times greater than cell growth in a batch fermentation system. In a preferred embodiment, cell growth in a perfusion system is at least 2 times greater than cell growth in a batch fermentation system.

[0133] In yet another aspect, the process of perfusion fermentation is carried out until the bacterial cell culture has a cell density, as determined by optical density at 600 nm (OD), of at least 20.0, e.g., at least 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, or 60.0. In a preferred embodiment, the process of perfusion fermentation is carried out until the cell density is at least 20.0.

[0134] In yet another aspect, the cell density of the bacterial cell cultures in the perfusion systems of the present invention, as determined by optical density at 600 nm (OD), can be at least 20.0, e.g., at least 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, or 60.0. In a preferred embodiment, the cell density is at least 20.0.

[0135] In another aspect of the invention, the concentration of polysaccharide in the perfusion system is at least 1.5 times, e.g., at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, or 3.5 times, higher than the concentration of polysaccharide in the batch fermentation system. In a preferred embodiment, the concentration of polysaccharide in the perfusion system is at least 2 times higher than the concentration of polysaccharide in the batch fermentation system.

[0136] For sialic acid-containing polysaccharides, such as GBS, the polysaccharide yield can be determined by measuring the concentration of sialic acid. Pelleted cells are digested by methods known in the art to release sialic acid from the cell-bound polysaccharides. The digest is assayed by anion exchange chromatography (AEX) using high-performance liquid chromatography (HPLC). The polysaccharide concentration is then determined by multiplying the sialic acid value by a repeating unit weight conversion factor. For example, the conversion factors for each serotype of GBS are as follows: Ia, Ib, and III = 3.24; II and V = 4.29; and IV = 3.77. Polysaccharide yield for S. pneumoniae or other encapsulated bacteria can be quantified by first releasing the capsular polysaccharide from the cell wall by treatment with detergents, such as sodium deoxycholate (DOC) or sodium N-laurylsarcosinate (NLS); acid treatment at high temperature; base treatment; and / or mechanical lysis. The released polysaccharides in the crude lysate are then assayed against certified standards using size exclusion chromatography (SEC) HPLC.

[0137] In one aspect, the perfusion fermentation process is carried out until the bacterial cell culture has a polysaccharide concentration, as determined by the concentration of sialic acid, of at least about 600 mg / L, e.g., at least about 650 mg / L; 700 mg / L; 750 mg / L; 800 mg / L; 850 mg / L; 900 mg / L; 950 mg / L; 1,000 mg / L; 1,500 mg / L; or 2,000 mg / L. In a preferred embodiment, the perfusion fermentation process is carried out until the polysaccharide concentration is at least about 600 mg / L.

[0138] In one aspect, the bacterial cell cultures produced by the perfusion systems of the present invention have a polysaccharide concentration, as determined by SEC HPLC, of ​​at least about 600 mg / L, e.g., at least about 650 mg / L; 700 mg / L; 750 mg / L; 800 mg / L; 850 mg / L; 900 mg / L; 950 mg / L; 1,000 mg / L; 1,500 mg / L; or 2,000 mg / L. In a preferred embodiment, the polysaccharide concentration is at least about 600 mg / L. [Example]

[0139] The following examples demonstrate some embodiments of the present invention. However, it should be understood that these examples are for illustrative purposes only and are not intended to be entirely definitive with respect to the conditions and scope of the present invention. When typical reaction conditions (e.g., temperature, reaction time, etc.) are given, it should be understood that conditions above and below the specified ranges can also be used, although generally to some degree less advantageous. Unless otherwise specified, all parts and percentages referred to herein are by weight, and all temperatures are expressed in degrees Celsius.

[0140] Additionally, the following examples were performed using standard techniques, which are well known and routine to those skilled in the art, but which have not otherwise been described in detail. As noted above, the following examples are presented for illustrative purposes and should not be construed as limiting the scope of the invention in any way.

[0141] Example 1 The defined medium of the present invention Applicant's proprietary defined medium for culturing mammalian cells ("R17") was modified for growing Streptococcus pneumoniae to create "modified AS3" medium (also referred to as "mAS3"). Modified AS3 medium was formulated using the components in Table 3 below. Media prepared for serotypes 4, 5, 6A, 6B, 14, and 23F were formulated with 30 g / L dextrose and 0.6 g / L magnesium sulfate. In the case of serotypes 1, 3, 6B, 7F, 9V, 18C, 19A, and 19F, 30 g / L dextrose was added to the medium in the reactor.

[0142] [Table 3]

[0143] A complete description of the amino acid, vitamin and salt composition in the R17 powder is provided in Table 4 below.

[0144] [Table 4]

[0145] Batch fermentations were carried out in 2 L bioreactors with temperature controlled at 36°C and pH controlled at 7.0 using NaOH as a base titrant. Fermenters were inactivated with an overlay of N2 for serotypes 1, 3, 4, 6A, 6B, 7F, 9V, 18C, 19A, and 19F, with air for serotypes 6B(2), 14, and 23F, and no overlay for serotype 5. Fermentations were agitated at 200 RPM. The results are shown in Table 5 below.

[0146] [Table 5]

[0147] Thirteen S. pneumoniae serotypes were successfully grown in batch culture using chemically defined modified AS3 medium.

[0148] Example 2 Comparison of commercially available defined media with the defined media of the present invention Eagle's Minimum Essential Medium (EMEM) is a commercially available defined cell culture medium that was tested against R17 using various GBS serotypes. Each formulation was modified from the label instructions to match the sodium bicarbonate requirements of anaerobically grown GBS. Each medium was also supplemented with a high concentration of glucose to support higher cell densities achievable in bacterial cultures. The formulation for modified EMEM for bacterial use ("EMEM for bacteria") is shown below: 20.2 g / L EMEM powder, 1.17 g / L L-glutamine, 0.84 g / L sodium bicarbonate, and 80 g / L glucose. For the growth of GBS, the composition of GBS mAS3 (as described in Example 1) mAS3 was custom tailored (hereafter "GBS mAS3") and was as follows: 0.21 g / L L-cysteine ​​HCl (instead of 300 mM acidic cystine stock solution), 2 mL / L trace element E (instead of 1 mL / L), 1000x, 0.84 g / L sodium bicarbonate, and 80 g / L glucose (instead of 25 g / L dextrose anhydrous).

[0149] Fermentations were carried out at a 10 L bioreactor scale, with the temperature controlled at 37°C and the pH controlled at 7.0 using NaOH as the base titrant. The fermentor was inert with an N2 overlay at 0.1 vvm relative to the batch volume, and the fermenter was thoroughly agitated and stirred to achieve a kLa of 1 / hr. The results are shown in Table 6 below.

[0150] [Table 6]

[0151] GBS mAS3 medium showed a striking advantage over bacterial EMEM medium in both growth and polysaccharide concentration.

[0152] Example 3 Comparison of complex media with the defined media of the present invention A comparison was made between modified AS3 medium as described in Example 1 and a soy hydrolysate-based complex medium (BPDv3) for various serotypes of S. pneumoniae. BPDv3 consisted of 28 g / L HYPEP1510 (Kerry Group Services Ltd.), 54 g / L glucose, 3.5 g / L NaCl, 0.7 g / L KH2PO4, 0.0182 g / L CaCl2·2H2O, 1 g / L MgSO4·7H2O, 0.84 g / L NaHCO3, 3 g / L ammonium chloride, 0.25 g / L uridine, 0.25 g / L adenosine, 0.03 g / L niacinamide, 0.03 g / L pyridoxine HCl, 0.0075 g / L pantothenic acid, and 0.003 g / L PABA. The medium for serotype 12F was supplemented with 1 g / L monosodium glutamate, and the medium for serotype 8 was modified to contain 0.5 g / L ammonium chloride and 36 g / L glucose. Although no consistent improvement in polysaccharide titers was obtained in modified AS3 medium compared to complex media, modified AS3 medium showed improved growth in almost all serotypes (see Table 7).

[0153] [Table 7]

[0154] Example 4 Amino acid consumption Analysis of amino acid consumption during the course of GBS serotype III fermentation was performed to determine whether amino acids were depleted. Analysis of amino acid concentrations in GBS mAS3 before inoculation and at harvest (as described in Example 2) is presented in Table 8.

[0155] [Table 8]

[0156] Although predicted required amino acids were not depleted, four amino acids for which S. agalactiae is likely prototrophic were depleted. Arginine, glycine, and serine were depleted below the limit of quantification. Cysteine, which is difficult to measure by HPLC methods, was not detected at any sample collection time. All other amino acids were still in excess at collection time.

[0157] GBS mAS3 medium was then supplemented with these four depleted amino acids at 4x the concentration of the base powder R17 formulation (16mM arg, 1.6mM cys, 6mM gly, and 40mM ser) in fermentations of various GBS serotypes. The results are shown in Table 9 below.

[0158] [Table 9]

[0159] Significant improvements in growth were observed for all six serotypes tested using CGRS-supplemented media. Although polysaccharide titers were not increased for all serotypes, the improved growth encouraged further testing.

[0160] Example 5 Further analysis of depleted amino acids The importance of each of the depleted amino acids to improved growth was evaluated in experiments where each of these four was sequentially removed from the medium using GBS serotype V as a model. Glycine was unexpectedly found to be the only one to contribute to improved growth (see Table 10).

[0161] [Table 10]

[0162] This was confirmed in a follow-up study in which each of these four amino acids was individually supplemented, again using GBS serotype V as a model, and this study confirmed that glycine was the only amino acid of the four depleted amino acids that improved growth and polysaccharide production (see Table 11).

[0163] [Table 11]

[0164] The importance of adding glycine as the only supplement was then tested in several serotypes of GBS. The performance of GBS mAS3, GBS mAS3 supplemented with all four amino acids, and GBS mAS3 supplemented with glycine only was compared. The results are shown in Table 12 below.

[0165] [Table 12]

[0166] In general, supplementation with glycine alone was sufficient to improve growth, and the improvement was similar to that achieved with supplementation with all four amino acids. Surprisingly, however, supplementation with glycine alone resulted in higher polysaccharide titers than GBS mAS3 and supplementation with all four amino acids.

[0167] Example 6 Comparison of glycine concentrations Given the unexpectedly high polysaccharide production associated with the addition of glycine alone, an experiment was conducted to determine whether the addition of 6 mM glycine to the GBS mAS3 formulation would result in maximum growth and polysaccharide titer. In this experiment, GBS serotype V was used as a model to compare the addition of 0.15 mM to 123.2 mM glycine. The data in Table 13 below demonstrate that the addition of as little as 1.5 mM glycine or as much as 61.6 mM glycine supports the same improvement in polysaccharide titer as observed with the addition of 6 mM glycine.

[0168] [Table 13]

[0169] Example 7 Determination of non-essential components of GBS mAS3 medium To ensure an excess of carbon source throughout and at the end of the fermentation, the GBS mAS3 formulation and its glycine-containing induction formulations of Examples 5 and 6 contained 80 g / L of glucose. Generally, when growth and polysaccharide production stopped, approximately 30 g / L of glucose remained unconsumed (data not shown). Therefore, experiments were conducted to determine whether a more efficient medium could be obtained. Using GBS serotype V as a model, glycine-supplemented GBS mAS3 media with glucose concentrations of 80 g / L, 70 g / L, 60 g / L, and 50 g / L were tested. The data in Table 14 below show that a glucose concentration of 50 g / L neither produced residual glucose nor compromised polysaccharide titers.

[0170] [Table 14]

[0171] Similarly, the importance of all amino acids and salts added to R17 powder was investigated by removing each one individually in a dropout experiment. This study was performed with GBS serotype V in glycine-supplemented GBS mAS3 medium. The data in Table 15 below show that tyrosine, glutamine, and cysteine ​​are essential for growth, while asparagine is not, and all salts are nonessential.

[0172] [Table 15]

[0173] Example 8 Vitamin and salt / trace element consumption Residual vitamins and salts / trace elements were assessed relative to starting concentrations using GBS serotype III as a model in glycine-supplemented GBS mAS3 medium. A total of 13 vitamins were examined: biotin, choline cyanocobalamin, folic acid, niacin, niacinamide, nicotinamide, p-aminobenzoic acid, pantothenic acid, pyridoxal, pyridoxamine, pyridoxine, riboflavin, and thiamine. Twelve amino acids showed no significant changes in concentration during fermentation. Niacinamide was found to be depleted to zero during the course of fermentation, but the accompanying accumulation of niacin would indicate that this vitamin family was not depleted (data not shown).

[0174] Thirty-two salts and trace elements were analyzed. Eighteen of these were below the detection limit. These 18 elements were: silver, aluminum, arsenic, beryllium, cadmium, chromium, copper, mercury, lithium, manganese, nickel, lead, rubidium, selenium, tin, titanium, thallium, and vanadium. Twelve of the 14 detectable salts and trace elements showed no substantial decrease in concentration from initial inoculation of the medium to post-harvest (see Table 16). Two elements showed a decrease in concentration: phosphorous acid and potassium. While a decrease in phosphorous acid concentration was expected because it is consumed for cell growth, phosphorous acid is not growth-limiting because it remained in excess at harvest. However, the decrease in potassium concentration was unexpected.

[0175] [Table 16]

[0176] These data led to two studies, this time examining the effect of adding an additional two-fold amount of potassium chloride (0.6 g / L added to 0.31 g / L R17 powder) to glycine-supplemented GBS mAS3 medium in fermentations of GBS serotype III. The results, shown in Table 17, indicate that increasing the KCl concentration was beneficial to polysaccharide titer, independent of growth.

[0177] [Table 17]

[0178] In further studies, a more complete range of KCl concentrations (0.03 g / L to 24 g / L added to the 0.31 g / L KCl contained in the basal powder R17) was examined for their effect on growth and polysaccharide synthesis. The results, shown in Table 18, indicate that KCl concentrations of 0.3 to 24 g / L added to the R17 powder resulted in improved growth and polysaccharide production.

[0179] [Table 18]

[0180] Example 9 mAS3opt50 medium formulation A medium was constructed ("mAS3opt50") by incorporating increased glycine and KCl, reduced glucose concentrations, and deletion of magnesium, asparagine, and NaCl in GBS mAS3 medium. The new formulation was tested against GBS mAS3 medium for six GBS serotypes. As shown in Table 19, the reformulated medium resulted in substantial improvements in growth and associated polysaccharide titers.

[0181] [Table 19]

[0182] Example 10 Contribution of glycine and KCl to polysaccharide yield in mAS3opt50 Using a dropout approach, the importance of glycine and KCl supplementation on polysaccharide yield was demonstrated in mAS3opt50 medium. The data shown in Table 20 clearly show that both are important in supporting high yields.

[0183] [Table 20]

[0184] Example 11 Comparison of mAS3opt50 with complex medium and complex medium supplemented with yeast extract The starting complex medium ("HP") was a soy hydrolysate-based formulation: 28 g / L HYPEP1510 (Kerry Group Services Ltd.), 3.5 g / L NaCl, 0.7 g / L KH2PO4, 0.0182 g / L CaCl2·2H2O, 1 g / L MgSO4·7H2O, 0.84 g / L NaHCO3, and 80 g / L glucose. Fermentation of GBS serotype 6 in this medium resulted in substantially lower growth and titers than in mAS3opt50. Therefore, HP was supplemented with 10 g / L of ultrafiltered yeast extract, AMBERFERM5902 (Sensient Technologies Corp.) ("HPYE"). HPYE medium had substantially improved cell density and polysaccharide titers compared to HP medium. HPYE showed increased growth compared to mAS3opt50 in almost all serotypes, while polysaccharide titers in HPYE medium were slightly lower. All data are shown in Table 21 below.

[0185] [Table 21]

[0186] Example 12 Increasing or decreasing the amount of yeast extract in complex media Increasing or decreasing the amount of yeast extract was performed to determine the concentration of supplemented yeast extract that would result in optimal growth and polysaccharide production. Using GBS serotype V as a model, the effect of yeast extract supplementation was determined using AMBERFERM5902 (Sensient Technologies Corp.) at 0 g / L, 2.5 g / L, 5 g / L, 10 g / L, 20 g / L, and 40 g / L. As shown in Table 22, these data indicated that supplementation with as little as 2.5 g / L of yeast was sufficient to stimulate growth and capsular polysaccharide production. However, the optimal concentration of supplemented yeast extract was 10 g / L, since adding higher amounts did not provide any additional benefit.

[0187] [Table 22]

[0188] Example 13 Supplementing defined media with yeast extract Given the positive impact of yeast extract supplementation on polysaccharide titers in complex media, experiments were conducted to examine supplementation of GBS mAS3 with AMBERFERM5902 (Sensient Technologies Corp.). GBS mAS3 supplemented with ultrafiltered yeast extract ("R17YE") was compared to GBS mAS3 and mAS3opt50. Supplementation of R17 with yeast extract dramatically improved polysaccharide titers compared to both GBS mAS3 and mAS3opt50 (see Table 23).

[0189] [Table 23]

[0190] A study was then conducted to compare supplementation with various concentrations of ultrafiltered yeast extract with various concentrations of a commercially available "synthetic" yeast extract (BD RECHARGE) from BD Biosciences. GBS serotype V was used as a model. The data, shown in Table 24, indicate that 20 g / L of yeast extract (whether ultrafiltered or synthetic) results in improved growth, but without a corresponding increase in polysaccharide titer. Supplementation with synthetic yeast extract improves growth over the GBS mAS3 control, but does not result in the maximum titer obtained with ultrafiltered yeast extract.

[0191] [Table 24]

[0192] Example 14 Analysis of fermentations with a constant glucose feed Using various GBS serotypes as models, a constant glucose feed was investigated for its effect on supporting polysaccharide titers using mAS3opt50 medium. Comparison of a 50 g / L glucose batch with a glucose-fed fermentation (10 g / L glucose batch, with the remaining 40 g / L fed at a constant rate over 7 hours starting at 3-4 hours EFT) showed comparable growth and polysaccharide titers across all serotypes (see Table 25). Other fermentation control parameters were those presented in Example 2.

[0193] [Table 25]

[0194] Example 15 Glucose fed-batch fermentation using HPYE and GBS mAS3 media Fed-batch fermentation was also investigated for HPYE and GBS mAS3 media using GBS serotype V as a model. Fermentation was initiated with a 10 g / L glucose batch, followed by a 70 g / L glucose feed over 5 hours 3-4 hours EFT. Fermentations were otherwise formulated as described in Example 2. The data presented in Table 26 show that the fed-batch approach results in roughly equivalent productivity compared to the batch approach for GBS mAS3. In HPYE, the fed-batch approach supports polysaccharide production, although productivity is slightly lower than batch.

[0195] [Table 26]

[0196] Example 16 perfusion fermentation Perfusion experiments were performed on 13 serotypes of S. pneumoniae using the modified AS3 medium described in Example 1. The medium was inoculated and cultured in a 2 L bioreactor in batch mode to obtain an OD 600 The culture was run for approximately 4-10 hours until a VV reached 3-7. The culture was then circulated through the perfusion system, removing spent medium and waste products and maintaining the culture volume by introducing fresh medium. Perfusion was initiated at an initial rate of 0.13 VVH and gradually increased to 0.80 VVH over 3-5 hours, at which point the perfusion batch was terminated. The data, shown below in Table 27, demonstrate a significant increase in biomass levels and a corresponding increase in polysaccharide production compared to the batch fermentation of Example 1.

[0197] [Table 27]

[0198] Example 17 Comparison of perfusion and batch fermentation methods in three different media Perfusion experiments were performed using GBS mAS3 or HPYE as the base medium. 1x medium (containing 0.5x glucose) was inoculated and run (5 L working volume) in batch mode for approximately 3 hours. When the OD approached 1-5 OD, perfusion with 0.5x medium was initiated at an initial rate of 0.13 VVH for approximately 1 hour. The rate was increased to 1.20 VVH over 6-7 hours, at which point the perfusion batch was terminated. Data for GBS mAS3 perfusion, shown below in Table 28, demonstrate an approximately 1.4- to 2-fold increase in cell density and concomitant increase in polysaccharide titer compared to batch mode.

[0199] [Table 28]

[0200] Data for perfusion based on HPYE complex medium are shown below in Table 29. In general, perfusion resulted in a greater than 2-fold increase in cell density and a 2-3.5-fold improvement in polysaccharide titer compared to batch.

[0201] [Table 29]

[0202] Perfusion in mAS3opt50-based medium was also performed using serotype IV as a model. The OD600 obtained at harvest was 16.7, with a polysaccharide production of 667 mg / L. In comparison, batch fermentation with mAS3opt50, presented in Example 8, resulted in a cell density of 10.4 and a polysaccharide production of 360 mg / L, demonstrating an approximately 1.9-fold improvement in productivity.

[0203] In summary, perfusion fermentation resulted in an approximately 2-fold or better increase in polysaccharide productivity compared to batch performance in all three media utilized.

[0204] Aspects of the present invention Additional embodiments of the present invention are described in the following clauses. C1. A medium for culturing polysaccharide-producing bacterial cells, the medium comprising a plant hydrolysate, a yeast extract and a carbon source. C2. The medium of C1, wherein the plant hydrolysate is a soybean hydrolysate. C3. The medium of C2, wherein the soy hydrolysate is selected from the group consisting of HYPEP1510 (Kerry Group Services Ltd.), HYPEP4601 (Kerry Group Services Ltd.), HYPEP5603 (Kerry Group Services Ltd.), HY-SOY (Kerry Group Services Ltd.), AMI-SOY (Kerry Group Services Ltd.), NZ-SOY (Kerry Group Services Ltd.), NZ-SOY BL4 (Kerry Group Services Ltd.), NZ-SOY BL7 (Kerry Group Services Ltd.), SHEFTONE D (Kerry Group Services Ltd.), SE50M, SE50MK, soy peptone, BACTO soytone (Difco Laboratories Inc.), NUTRISOY2207 (ADM), NUTRISOY (ADM), NUTRISOY flour (ADM), and soybean meal. C4. The medium described in C3, wherein the soy hydrolysate is HYPEP1510 (Kerry Group Services Ltd.). C5. The medium of any one of C1 to C4, wherein the concentration of the plant hydrolysate is between about 5 g / L and about 75 g / L. C6. The medium of C5, wherein the concentration of the plant hydrolysate is between about 10 g / L and about 50 g / L. C7. The medium of C6, wherein the concentration of the plant hydrolysate is about 28 g / L. C8. The medium of any one of C1 to C7, wherein the yeast extract is a yeast autolysate, an ultrafiltered yeast extract, or a synthetic yeast extract. C9. The medium of C8, wherein the yeast extract is an ultrafiltered yeast extract. C10. The medium described in C9, wherein the ultrafiltered yeast extract is AMBERFERM5902 (Sensient Technologies Corp.), BD DIFCO (BD Biosciences), HYPEP YE (Kerry Group Services Ltd.), ULTRAPEP YE (Kerry Group Services Ltd.), HY-YEST412 (Kerry Group Services Ltd.), HY-YEST441 (Kerry Group Services Ltd.), HY-YEST444 (Kerry Group Services Ltd.), HY-YEST455 (Kerry Group Services Ltd.), or HY-YEST504 (Kerry Group Services Ltd.). C11. The medium of any one of C1 to C10, wherein the concentration of yeast extract is between about 1 g / L and about 50 g / L. C12. The medium of C11, wherein the concentration of yeast extract is between about 5 g / L and about 25 g / L. C13. The medium of C12, wherein the concentration of yeast extract is about 10 g / L. C14. The medium of any one of C1 to C13, wherein the carbon source is selected from the group consisting of glucose, dextrose, mannitol, lactose, sucrose, fructose, galactose, raffinose, xylose and mannose. C15. The medium of C14, wherein the carbon source is glucose. C16. The medium of any one of C1 to C15, wherein the concentration of the carbon source is between about 25 g / L and about 100 g / L. C17. The medium of C16, wherein the concentration of the carbon source is between about 50 g / L and about 90 g / L. C18. The medium of C17, wherein the concentration of the carbon source is about 80 g / L. C19. The medium of any one of C1 to C18, comprising soy hydrolysate, ultrafiltered yeast extract, and glucose. C20. The medium of any one of C1 to C19, further comprising a phosphate-containing component. C21. The medium according to C20, wherein the phosphate-containing component is Na2HPO4, K2HPO4, or KH2PO4. C22. The medium of any one of C1 to C21, further comprising at least one amino acid, vitamin, nucleoside, or inorganic salt. C23. A medium for culturing polysaccharide-producing bacterial cells, the medium having a total concentration of amino acids greater than about 50 mM. C24. The medium of C23, comprising glycine at a total concentration of between about 1.5 mM and about 60.0 mM. C25. The medium according to C24, wherein the total concentration of glycine is between about 5.0 mM and about 15.0 mM. C26. The medium of C25, wherein the total concentration of glycine is about 7.5 mM. C27. The medium of any one of C23 to C26, comprising arginine at a total concentration of between about 1.0 mM and about 30.0 mM. C28. The medium according to C27, wherein the total concentration of arginine is between about 1.0 mM and about 20.0 mM. C29. The medium of C28, wherein the total concentration of arginine is about 4.0 mM. C30. The medium of any one of C23 to C29, comprising cysteine ​​at a total concentration of between about 0.1 mM and about 5.0 mM. C31. The medium according to C30, wherein the total concentration of cysteine ​​is between about 0.1 mM and about 3.5 mM. C32. The medium of C31, wherein the total concentration of cysteine ​​is about 0.4 mM. C33. The medium of any one of C23 to C32, comprising serine at a total concentration of between about 5.0 mM and about 75.0 mM. C34. The medium according to C33, wherein the total concentration of serine is between about 5.0 mM and about 15.0 mM. C35. The medium of C34, wherein the total concentration of serine is about 7.5 mM or about 10 mM. C36. The medium of any one of C23 to C35, comprising glutamine at a total concentration of between about 1.0 mM and about 30.0 mM. C37. The medium according to C36, wherein the total concentration of glutamine is between about 1.0 mM and about 20.0 mM. C38. The medium of C37, wherein the total concentration of glutamine is about 4.0 mM. C39. The medium of any one of C23 to C38, comprising tyrosine at a total concentration of between about 0.1 mM and about 5.0 mM. C40. The medium of C39, wherein the total concentration of tyrosine is between about 1.0 mM and about 3.5 mM. C41. The medium of C40, wherein the total concentration of tyrosine is about 2.9 mM or about 3.0 mM. C42. The medium of any one of C23 to C41, comprising asparagine at a total concentration of between about 5.0 mM and about 50.0 mM. C43. The medium of C42, wherein the total concentration of asparagine is between about 10.0 mM and about 30.0 mM. C44. The medium of C43, wherein the total concentration of asparagine is about 20.0 mM. C45. The medium of any one of C23 to C41, which does not contain asparagine. C46. The medium of any one of C23 to C45, further comprising a potassium salt. C47. The medium of C46, ​​wherein the potassium salt is potassium chloride or potassium sulfate. C48. The medium of any one of C46 to C47, wherein the total concentration of potassium salts is between about 0.1 g / L and about 25 g / L. C49. The medium of C48, wherein the total concentration of potassium salts is between about 0.2 g / L and about 1.25 g / L. C50. The medium of C49, wherein the total concentration of potassium salts is about 0.9 g / L. C51. The medium of any one of C23 to C50, further comprising a carbon source. C52. The medium of C51, wherein the carbon source is selected from the group consisting of glucose, dextrose, mannitol, lactose, sucrose, fructose, galactose, raffinose, xylose, and mannose. C53. The medium of C52, wherein the carbon source is glucose. C54. The medium of any one of C51 to C53, comprising a carbon source at a total concentration of between about 25 g / L and about 100 g / L. C55. The medium of C54, wherein the total concentration of carbon sources is between about 25 g / L and about 80 g / L. C56. The medium of C55, wherein the total concentration of carbon sources is about 50 g / L. C57. The medium of any one of C23 to C56, further comprising sodium bicarbonate. C58. The medium of C57, comprising sodium bicarbonate at a concentration of between about 0.1 g / L and about 20 g / L. C59. The medium according to C58, wherein the concentration of sodium bicarbonate is between about 0.5 g / L and about 1.0 g / L. C60. The medium of C59, wherein the concentration of sodium bicarbonate is about 0.84 g / L. C61. The medium of any one of C23 to C60, further comprising yeast extract. C62. The medium of C61, wherein the yeast extract is selected from the group consisting of yeast autolysate, ultrafiltered yeast extract, and synthetic yeast extract. C63. The medium of C62, wherein the yeast extract is an ultrafiltered yeast extract. C64. The medium of C63, wherein the ultrafiltered yeast extract is AMBERFERM5902 (Sensient Technologies Corp.), BD DIFCO (BD Biosciences), HYPEP YE (Kerry Group Services Ltd.), ULTRAPEP YE (Kerry Group Services Ltd.), HY-YEST412 (Kerry Group Services Ltd.), HY-YEST441 (Kerry Group Services Ltd.), HY-YEST444 (Kerry Group Services Ltd.), HY-YEST455 (Kerry Group Services Ltd.), or HY-YEST504 (Kerry Group Services Ltd.). C65. The medium of any one of C61 to C64, wherein the concentration of yeast extract is between about 1 g / L and about 50 g / L. C66. The medium of C65, wherein the concentration of yeast extract is between about 5 g / L and about 25 g / L. C67. The medium of C66, wherein the concentration of yeast extract is about 10 g / L. C68. The medium of any one of C23 to C67, comprising at least about 50 mM amino acids; a potassium salt, a carbon source, and optionally yeast extract. C69. The medium of C68, comprising at least about 50 mM amino acids, between about 5.0 mM and about 15.0 mM glycine, between about 0.2 g / L and about 1.25 g / L of potassium salt, between about 25 g / L and about 80 g / L of carbon source, and between about 5 g / L and about 25 g / L of yeast extract. C70. The medium of C69, comprising at least about 60 mM amino acids, about 7.5 mM glycine, about 0.9 g / L potassium chloride, 50 g / L glucose, and about 10 g / L ultrafiltered yeast extract. C71. A method for growing polysaccharide-producing bacteria, comprising the steps of: a) adding to a bioreactor the medium of any one of C1 to C70; b) inoculating the medium with polysaccharide-producing bacteria; and c) growing the bacteria by fermentation, wherein said growing comprises the constant addition of nutrients to the medium. C72. The method of growing according to C71, wherein the nutrient is a carbon source. C73. The growth method of C72, wherein the carbon source is glucose. C74. The method of any one of C71 to C73, wherein the grown bacteria exhibits a cell density of at least 9.0. C75. The method of any one of C71 to C74, wherein the grown bacteria exhibits a polysaccharide concentration of at least about 250 mg / L. C76. The method of any one of C71 to C75, wherein the polysaccharide-producing bacteria 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. C77. A method for growing polysaccharide-producing bacteria, comprising: a) adding to a bioreactor the medium of any one of C1 to C70; b) inoculating the medium with polysaccharide-producing bacteria; and c) growing the bacteria by perfusion, wherein growing comprises (i) removing spent medium from the culture, (ii) adding fresh medium, and (iii) retaining the bacteria. C78. The growth method of C77, wherein the perfusion rate is between about 0.07 VVH and about 2.00 VVH. C79. The growth method of C78, ​​wherein the perfusion rate is between about 0.67 VVH and about 1.33 VVH. C80. The growth method of C79, wherein the perfusion rate is about 1.20 VVH. C81. The growth method of C77, wherein the perfusion rate is variable. C82. The method of growing of C81, wherein perfusion is initiated at a first rate and the rate is increased to a second rate. C83. The method of growing of C81, wherein perfusion is initiated at a first rate and the rate is decreased to a second rate. C84. The growth method of any one of C77 to C83, wherein the duration of perfusion is between about 1 hour and about 15 hours. C85. The growth method according to C84, wherein the duration of perfusion is between about 1 hour and about 10 hours. C86. The growth method of C85, wherein the duration of perfusion is about 7 hours. C87. The method of any one of C77 to C86, wherein cell growth of the grown bacteria is at least two-fold greater than cell growth in a batch fermentation system. C88. The method of any one of C77 to C87, wherein the grown bacteria reaches a cell density of at least 20.0. C89. The method of any one of C77 to C88, wherein the grown bacteria reaches a polysaccharide concentration of at least about 600 mg / L. C90. The method of any one of C77 to C89, wherein the polysaccharide-producing bacteria 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.

Claims

1. Polysaccharide-producing Streptococcus pneumoniae pneumoniae or Streptococcus agalactiae agalactiae), the medium comprising a soybean hydrolysate, vitamins, nucleosides, and a carbon source, wherein the vitamins include niacinamide, p-aminobenzoic acid, pantothenic acid, and pyridoxine HCl, the nucleosides include adenosine and uridine, and the carbon source is selected from the group consisting of glucose, dextrose, mannitol, lactose, sucrose, fructose, galactose, raffinose, xylose, and mannose.

2. The culture medium described in claim 1, wherein the soybean hydrolysate is 5 g / L to 75 g / L.

3. 3. The medium according to claim 1, wherein the concentration of the vitamin is 0.001 g / L to 1 g / L.

4. The medium according to any one of claims 1 to 3, wherein the concentration of the nucleoside is 0.1 g / L to 1 g / L.

5. 5. The medium according to claim 1, wherein the carbon source is glucose.

6. 6. The medium according to any one of claims 1 to 5, wherein the concentration of the carbon source is between 25 g / L and 100 g / L.

7. The medium according to any one of claims 1 to 6, further comprising salt.

8. 8. The medium of claim 7, wherein the salt is selected from the group consisting of ammonium chloride, calcium chloride, magnesium sulfide, monosodium glutamate, potassium chloride, potassium sulfide, and sodium chloride.

9. 9. The medium of claim 8, wherein the salt is ammonium chloride.

10. 9. The medium of claim 8, wherein the salt is calcium chloride.

11. 9. The medium of claim 8, wherein the salt is magnesium sulfide.

12. 9. The medium of claim 8, wherein the salt is sodium chloride.

13. 9. The medium of claim 8, wherein the salt is a mixture of salts comprising ammonium chloride, calcium chloride, magnesium sulfate, and sodium chloride.

14. 9. The medium of claim 8, wherein the salt is a mixture of salts comprising ammonium chloride, calcium chloride, magnesium sulfate, and sodium chloride.

15. 15. The medium of claim 13 or 14, wherein the mixture of salts further comprises monosodium glutamate.

16. 16. The medium of claim 1, further comprising a phosphate-containing component.

17. The phosphate-containing component is Na ２ HPO ４ , K. ２ HPO ４ or KH ２ P.O. ４ The medium according to claim 16,

18. 18. The medium of claim 1, further comprising sodium bicarbonate.

19. 2. The medium according to claim 1, wherein the vitamins are a mixture of vitamins including niacinamide, p-aminobenzoic acid, pantothenic acid, and pyridoxine HCl, the nucleosides are a mixture of nucleosides including adenosine and uridine, and the carbon source is glucose, and the medium further contains a mixture of salts including ammonium chloride, calcium chloride, magnesium sulfide, and sodium chloride, and KH ２ P.O. ４ and a phosphate-containing component including sodium bicarbonate.

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