WO2016207744A1

WO2016207744A1 - Improved process for production and purification of capsular pneumococcal polysaccharide from streptococcus pneumoniae and method for producing conjugate vaccine composition using said polysaccharide

Info

Publication number: WO2016207744A1
Application number: PCT/IB2016/053059
Authority: WO
Inventors: Ganesh Kumraj; Jainender JAIN; Davender Kumar BHATI; Ashok Kumar; Vijay Kumar
Original assignee: Biobridge Healthcare Solutions Pvt. Ltd.
Priority date: 2015-06-26
Filing date: 2016-05-25
Publication date: 2016-12-29

Abstract

The present disclosure provides a culture medium capable of being used for clonal growth of Streptococcus pneumoniae to produce capsular pneumococcal polysaccharide for vaccine production, wherein the culture medium can include KH₂PO₄, NaHCO₃, soya peptone, yeast extract, MgSO₄, NaCl, CaCl₂, glucose and L-cysteine or its salts. The present disclosure further provides a method of fermentation of Streptococcus pneumoniae to produce capsular pneumococcal polysaccharide.

Description

IMPROVED PROCESS FOR PRODUCTION AND PURIFICATION OF CAPSULAR PNEUMOCOCCAL POLYSACCHARIDE FROM STREPTOCOCCUS PNEUMONIAEAND METHOD FOR PRODUCING CONJUGATE VACCINE COMPOSITION USING SAID POLYSACCHARIDE

This is an application for patent of addition for which the main application is 349/MUM/2014 filed on 31/01/2014.

FIELD OF THE INVENTION

[0001] The present disclosure relates to production and purification of capsular pneumococcal polysaccharide from streptococcus pneumoniae of serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F & 23F, and method for producing conjugate vaccine composition using said polysaccharide.

BACKGROUND OF THE INVENTION

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Children less than 2 years of age do not mount an immune response to most polysaccharide vaccines, so it has been necessary to render the polysaccharides immunogenic by chemical conjugation to a protein carrier. Coupling the polysaccharide, a T-independent antigen, to a protein, a T-dependent antigen, confers upon the polysaccharide the properties of T dependency including isotype switching, affinity maturation, and memory induction.

[0004] The effect of the carrier-protein dosage on the humoral response to the protem itself has also proven to be multifaceted. In human infants it was reported that increasing the dosage of a tetravalent tetanus toxoid conjugate resulted in a decreased response to the tetanus carrier. Classical analysis of these effects of combination vaccines have been described as carrier induced epitopic suppression, which is not fully understood, but believed to result from an excess amount of carrier protein (Fattom, Vaccine 17: 126 (1999)). This appears to result in competition for Th-cells, by the B-cells to the carrier protein, and B-cells to the polysaccharide. If the B-cells to the carrier protein predominate, there are not enough Th-cells available to provide the necessary help for the B-cells specific to the polysaccharide. However, the observed immunological effects have been inconsistent, with the total amount of carrier protein in some instances increasing the immune response, and in other cases diminishing the immune response. Hence there remain technical difficulties in combining multiple polysaccharide conjugates into a single, efficacious, vaccine formulation (Jedrzejas MJ. Pneumococcal virulence factors: structure and function. Microbiology and Molecular Biology Reviews, 2001, 65: 187-207)

[0005] Streptococcus pneumoniae (S. pneumoniae) is a normal inhabitant of the human upper respiratory tract. The bacterium can cause pneumonia, usually of the lobar type, paranasal sinusitis and otitis media, or meningitis, which is usually secondary to one of the former infections. It also causes osteomyelitis, septic arthritis, endocarditis, peritonitis, cellulitis and brain abscesses. Streptococcus pneumoniae is currently the leading cause of invasive bacterial disease in children and the elderly. Streptococcus pneumoniae is known in medical microbiology as the pneumococcus, referring to its morphology and its consistent involvement in pneumococcal pneumonia. (Musher DM. Infections caused by Streptococcus pneumoniae: clinical spectrum, pathogenesis, immunity, and treatment. Clinical Infectious Diseases, 1992, 14:801-807)

[0006] S. pneumoniae is an important cause of otitis media, meningitis, bacteremia and pneumonia, and a leading cause of fatal infections in the elderly and persons with underlying medical conditions, such as pulmonary disease, liver disease, alcoholism, sickle cell, cerebrospinal fluid leaks, acquired immune deficiency syndrome (AIDS), and patients undergoing immunosuppressive therapy. It is also a leading cause of morbidity in young children. The most severe pneumococcal infections involve invasive meningitis and bacteraemia infections.

[0007] 30% of pneumococcal isolates are resistant to penicillin. The increase in antimicrobial resistant pneumococcus further emphasizes the need for preventing pneumococcal infections.

(Sethi S, Murphy TF. Bacterial infection in chronic obstructive pulmonary disease in 2000: a state-of-the-art review. ClmMicrobiol Rev. 2001 Apr;14(2):336-63).

[0008] Humans acquire pneumococci through aerosols or by direct contact. Pneumococci first colonize the upper airways and can remain in nasal mucosa for weeks or months. In most cases, this colonization results in no apparent infection. The organism carried in the nasopharynx can give rise to symptomatic sinusitis or middle ear infections. If pneumococci are aspirated into the lung, especially with food particles or mucus, they can cause pneumonia. Infections at these sites generally shed some pneumococci into the blood, where they can lead to sepsis, especially if they continue to be shed in large numbers from the original focus of infection. (Musher DM. Streptococcus pneumoniae. In Mandell GL, Bennett JE, Dolin R (eds). Principles and Practice of Infectious diseases (5th ed). New York, Churchill Livingstone, 2001 , p2128- 2147). Pneumococci in the blood can reach the brain where they can cause meningitis.

[0009] An attractive goal for streptococcal vaccination is to reduce carriage in the vaccinated populations and subsequently reduce the incidence of pneumococcal disease. There is speculation that a reduction in pneumococcal carriage rates by vaccination could reduce the incidence of the disease in non-vaccinated individuals as well as vaccinated individuals. This "herd immunity" induced by vaccination against upper respiratory bacterial pathogens has been observed using the Haemophilusinfluenzae type b conjugate vaccines. If a vaccine could prevent colonization by pneumococci such vaccine would be expected to prevent virtually all pneumococcal infections in the immunized patents. Since even immunized patients might acquire pneumococci from others, a vaccine that reduced carriage should reduce infections in immuno-compromised as well as uncompromised patients. It is generally accepted that immunity to S. pneumoniae can be mediated by specific antibodies against the polysaccharide capsule of the pneumococcus.

[0010] Pneumococcal capsular polysaccharides are responsible for its anti-phagocytic properties and inhibition of adherence to host cells, which is a critical step in carriage and possibly later aspects in the pathogenesis of disease. Hence the capsule of S. pneumoniae has long been recognized as the major virulence factor. There are over ninety known capsular sero types of S. pneumoniae, of which twenty-three account for about 95% of the disease (Whitney CG, et al. Effectiveness of seven-valent pneumococcal conjugate vaccine against invasive pneumococcal disease: a matched case-control study. Lancet, 2006, 368: 1495- 1502).

[0011] Culture of S. pneumoniaeto produce pneumococcal polysaccharides for vaccine production, particularly at the inoculum development stage, is typically performed in culture media containing complex bovine or ovine derived products such as peptones, gelatins, caseins, or blood derived products. S. pneumoniae are known to be fastidious, having nutritional requirements for choline [Rave et al. J. Bact, 40, 695-704 (1940)], vitamins [Adams et 1, J. Bact, 49 401-409 (1945)], and biotin [Bohonos and Sabborow, Arch. Biochem. 3, 257-259 (1943)]. Even where subsequent culture of bacteria in media free of complex bovine or ovine derived products has been achieved, initial development of the culture inoculum of S. pneumoniae has been accomplished in blood- containing or bovine/ovine derived product media.

[0012] Use of blood components or animal extracts in the culture media may pose a serious health hazard as the probability of contaminants like adventitious viruses, prions and mycoplasma is more, as they may get passed on to the substance produced using them. Further, the adsorption of blood and animal extracts derived proteins on the cell surface of the bacteria may also adversely affect the cell surface chemistry, which is very critical for in vitro microenvironment in numerous cell types.

[0013] Few animal component free culture media for S. pneumoniae are also known in the prior art. For example: a) ApplMicrobiolBiotechnol (2002) 59:713-717 (V.M. Goncalves, et al) discloses an animal component free medium for Streptococcus pneumoniae serotype 23F. Although an increased biomass production was obtained here, an improvement in polysaccharide production was not obtained using the reported media, b) J IndMicrobiolBiotechnol (2008) 35: 1441-1445 (C. Liberman, et al) discloses an animal-free medium for propagation of S. pneumoniae for pneumococcal whole cell vaccine preparation and not polysaccharide based vaccines. Further, exclusion of the blood or animal extracts leads to supplementing the media with number of other amino acids, vitamins etc. making the media expensive. Ideally, the culture medium should contain only essential components and should be easily prepared in a reproducible manner.

[0014] There is thus a need in the art for a new and improved culture medium that is safe, free of animal or blood products, highly economic and allows for large scale production of polysaccharide in high purity and yields. There is also a need in the art for an improved method of fermentation of S. pneumoniaeto produce capsular pneumococcal polysaccharide for vaccine production.

[0015] The present invention satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.

OBJECTS OF THE INVENTION

[0016] It is an object of the present disclosure to provide a novel culture medium capable of being used for clonal growth of Streptococcus pneumoniae to produce capsular pneumococcal polysaccharide.

[0017] It is a further object of the present disclosure to provide a culture medium for Streptococcus pneumoniaethat is devoid of bovine derived, ovine derived or crude blood derived products.

[0018] It is another object of the present disclosure to provide a culture medium that allows for effective production of capsular pneumococcal polysaccharide with high purity and yield. [0019] It is another object of the present disclosure to provide a method of fermentation of Streptococcus pneumoniae to produce capsular pneumococcal polysaccharide for vaccine production.

SUMMARY OF THE INVENTION

[0020] The present disclosure provides a culture medium capable of being used for clonal growth of Streptococcus pneumoniae to produce capsular pneumococcal polysaccharide for vaccine production.

[0021] According to embodiments of the present disclosure, the culture medium can include KH₂PO₄, NaHCC , soya peptone, yeast extract, MgS0₄, NaCl, CaCl₂, glucose and L-cysteine or its salts.

[0022] In an exemplary embodiment of the present disclosure, the culture medium can include KH₂P0₄, NaHC0₃, soya peptone, yeast extract, MgS0₄.7H₂0, NaCl, CaCl₂, D-glucose and hydrochloride salt of L-cysteine.

[0023] In a further exemplary embodiment of the present disclosure, the culture medium can be an aqueous solution including, on a per liter basis,0.1 to 2g of KH₂P0₄, 0.1 to 2g of NaHCCh, 1 to 50g of soya peptone, 1 to 30g of yeast extract, 0.01 to 2g of MgS0₄, 0.1 to 5g of NaCl, 0.01 to 2g of CaCl₂, 1 to 50g of glucose and 0.01 to 2g of L-cysteine.

[0024] In a more specific exemplary embodiment of the present disclosure, the culture medium can be an aqueous solution including, on a per liter basis, 0.5g of KH₂P0₄, 0.22g of NaHCC , 30g of soya peptone, 20g of yeast extract, 0.4g of MgS0₄.7H₂0, 2.5g of NaCl, O.Olg of CaCl₂, lOg of D-glucose and 0.15g of L-cysteine.HCl.

[0025] In an embodiment, the present disclosure provides a culture medium capable of being used for growth of Streptococcus pneumoniae of various serotypes which can be selected from 1 , 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

[0026] In another aspect, the present disclosure provides a method of fermentation of Streptococcus pneumoniae to produce capsular pneumococcal polysaccharide, wherein the method can include the steps of: (a) providing a culture medium comprising KH₂P0₄, NaHCC , soya peptone, yeast extract, MgS0₄, NaCl, CaCl₂, glucose and L-cysteine or its salts; (b) growing Streptococcus pneumoniae in said culture medium; (c) inactivating cells in culture broth; (d) recovering capsular pneumococcal polysaccharide from said culture medium; and (e) optionally purifying capsular pneumococcal polysaccharide obtained in step (d). [0027] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0029] Each of the appended claims defines a separate invention, which for infringement purposes is recognized as including equivalents to the various elements or limitations specified in the claims. Depending on the context, all references below to the "invention" may in some cases refer to certain specific embodiments only. In other cases it will be recognized that references to the "invention" will refer to subject matter recited in one or more, but not necessarily all, of the claims.

[0030] Unless the context requires otherwise, throughout the specification which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense that is as "including, but not limited to."

[0031] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0032] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.

[0033] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0034] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0035] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0036] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0037] The present disclosure provides a culture medium capable of being used for clonal growth of Streptococcus pneumoniae to produce capsular pneumococcal polysaccharide. The key aspect of the culture medium is the absence of blood components, bovine or ovine derived products and the ability of the culture medium to support growth of Streptococcus pneumoniae and thereby the production of capsular pneumococcal polysaccharides useful for vaccine production. [0038] According to embodiments of the present disclosure, the culture medium can include KH₂PO₄, NaHCC , soya peptone, yeast extract, MgS0₄, NaCl, CaCl₂, glucose and L-cysteine or its salts.

[0039] In an exemplary embodiment, MgS0₄ used to formulate the culture medium of the present disclosure can be hydrated such as, but not limited to,MgS0₄.7H₂0, glucose can be D- glucose and L-cysteine can be hydrochloride salt of L-cysteine.

[0040] In a further exemplary embodiment of the present disclosure, the culture medium can be an aqueous solution containing, on a per liter basis,0.1 to 2g of KH₂P0₄, 0.1 to 2g of NaHCCh, 1 to 50g of soya peptone, 1 to 30g of yeast extract, 0.01 to 2g of MgS0₄, 0.1 to 5g of NaCl, 0.01 to 2g of CaCl₂, 1 to 50g of glucose and 0.01 to 2g of L-cysteine.However, it is to be appreciated that the amounts/concentrations of components in the culture medium can be optimized or modified to meet desired performance characteristics.

[0041] In a more specific exemplary embodiment of the present disclosure, the culture medium can be an aqueous solution containing, on a per liter basis, 0.5g of KH₂P0₄, 0.22g of NaHCCh, 30g of soya peptone, 20g of yeast extract, 0.4g of MgS0₄.7H₂0, 2.5g of NaCl, O.Olg of CaCl₂, lOg of D-glucose and 0.15g of L-cysteine.HCl.

[0042] In an embodiment, the present disclosure provides a culture medium capable of being used for growth of Streptococcus pneumoniae of various serotypes which can be selected from 1 , 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. Fermentation process parameters may vary depending upon the serotype of Streptococcus pneumoniae being used for cultivation.

[0043] The culture medium of the present disclosure, by virtue of its low protein content, can prevent damages to microenvironment of cells, in vitro, which is one of the major factors affecting growth and production capacity of a microorganism.

[0044] In another aspect, the present disclosure provides a method of fermentation of Streptococcus pneumoniae to produce capsular pneumococcal polysaccharide, wherein the method can include the steps of: (a) providing a culture medium comprising KH₂P0₄, NaHCCh, soya peptone, yeast extract, MgS0₄, NaCl, CaCl₂, glucose and L-cysteine or its salts; (b) growing Streptococcus pneumoniae in said culture medium; (c) inactivating cells in culture broth; (d) recovering capsular pneumococcal polysaccharide from said culture medium; and (e) optionally purifying capsular pneumococcal polysaccharide obtained in step (d).

[0045] According to embodiments, the method of fermentation of Streptococcus pneumoniae to produce capsular pneumococcal polysaccharide can include a step of pre-culturing Streptococcus pneumoniae in the culture medium of the present disclosure to obtain a seed culture and subsequently fermenting the seed culture in main fermentation medium. The seed culture can be prepared by any methods known in the art. The culture may be grown under appropriate conditions until the optimum optical density is achieved.

[0046] According to embodiments of the present disclosure, the culture medium used in seed culture and in main fermentation may have identical or different composition.

[0047] In a more preferred embodiment, the main fermentation medium can be inoculated by a seed culture having, on a per liter basis, 0.5g of KH₂P0₄, 0.22g of NaHCC>3, 30g of soya peptone, 20g of yeast extract, 0.4g of MgS0₄.7H₂0, 2.5g of NaCl, O.Olg of CaCl₂, lOg of D- glucose and 0.15g of L-cysteine.HCl.

[0048] The seed culture can be inoculated into main fermentation medium with appropriate cultivation time. In main fermentation stage, a "steady state" condition with maximal polysaccharide production rate can be maintained for a long time by feeding of organic carbon sources in order to supply the nutrient demand.

[0049] In an exemplary embodiment, steady state conditions can be maintained in main fermentation stage by feeding aqueous solution of D-glucose.

[0050] Air can be sparged into fermentation medium for the purpose of providing oxygen for growth of culture. After fermentation is completed, formalin or other agents may be added to inactivate the culture. The inactivated culture may be subjected to treatment with sodium deoxycholate to effect polysaccharide release from the surface of cells. The culture may be harvested using centrifugation or filtration followed by concentration and diafiltration of the supernatant to extract pneumococcal polysaccharide.

[0051] In accordance with embodiments of the present disclosure, fermentation of Streptococcus pneumoniae may be done in the culture medium by batch, fed- batch or continuous fermentation.

EXAMPLES

[0052] The present invention is further explained in the form of following examples. However it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention. Example 1: Preparation of culture medium

[0053] Solution A was prepared by dissolving 30g of soya peptone and 20g of yeast extract in 1 liter of water under stirring, then it was sterilized at 121.1°C for 20 minutes. Solution B was prepared by dissolving 2.5 g of NaCl, 0.5g of KH₂P0_4, 0.4g ofMgS0₄.7H₂0, 0.01 g of CaCl_2, 0.15 g ofL-Cysteine.HCl in 1 liter of water under stirring, then it was sterilized at 121.1°C for 20 minutes. Solution C was prepared by dissolving lOg of D-glucose in 1 liter of water under stirring. Solution C was autoclaved at 121.1°C for 10 minutes and then satirized at 121.1°C for 10 minutes. Solution D was prepared by dissolving 0.22g of NaHCC in 1 liter of water under stirring, then it was autoclaved at 121.1°C for 10 minutes. Solutions A, B, C and D were mixed together and the pH of the resultant mixture was adjusted to 7.2-7.3using 2M NaOH to yield the culture medium.

Example 2: Preparation of seed culture

[0054] 30 ml - 40 ml of the culture medium as prepared in example 1 was transferred into a Erlenmeyer flask of 100 ml capacity and it was inoculated with 1 ml of frozen seed lot of Streptococcus pneumoniae strains. The cells were incubated for 8 - 10 hrs at 35°C in a humidified 5% C02 incubator. After completion of incubation period, purity was checked by gram staining and optical density at 600nm. When the optical density of the culture reaches 1.2 - 2.2 add 10ml of the seed@ 4% to the same media of 250 ml /1000 ml flask and incubate for 8 - 10 hrs at 35°C in a humidified 5% C02 incubator. After completion of incubation period, purity was checked by gram staining and optical density at 600nm is alsO checked. When the optical density of the culture reaches 1.5 - 2.5 pool the seed from all the three flasks and inoculate 750 ml @ 5% to the same media of 15 lit working volume of the fermenter.

Example 3: Preparation of feed for fermentation

[0055] 400g of D-glucose was dissolved in 1 liter of water under stirring and the resultant solution was autoclaved at 121.1 °C for 10 minutes.

Example 4: Fermentation of Streptococcus pneumoniae

[0056] The above seed culture was transferred into a bioreactor containing 15000 ml of the culture medium as prepared in example 1 for fermentation. The culture was inoculated at 5% inoculation rate. Each of Streptococcus pneumoniae of serotype 1, 3, 4, 5, 6 A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33 were grown in the culture medium.Process parameters used for fermentation of various serotypes of Streptococcus pneumoniae are provided in the below table 1. After incubation, samples were collected during growth for measuring optical density (OD) of the culture at 600nm. When the OD reached 2 -4 AU, feed (D-glucose solution as prepared in Ex.1) was initiated at the rate of 0.1ml/ 1/ min and gradually increased to and 0.2ml/ 1/ min. Feed was stopped when the cultivation reached stationary phase. The fermentation process was terminated once the desired OD (600nm) of the culture was achieved. Optical density for different serotypes of Streptococcus pneumoniae for batch termination is provided in the below table 2. The fermentation broth was sparged with air at the rate of 0.1 VVM for about lhour. Then, 13% solution of sodium deoxycholate (DOC) was added at the final concentration of 0.13% to the fermented broth. After DOC treatment, the culture was harvested in a sterilized bottle and centrifuged at 7500 rpm for 30 min. The supernatant was taken for further purification.

Table 1

Serotype of Streptococcus pH Agitation (RPM) Temp (°C)

pneumoniae

1 7.1 ± 1 80 ± 10 35

3 7.1 ± 1 80 ± 10 35

4 7.1 ± 1 80 ± 10 35

5 7.1 ± 1 80 ± 10 35

6A 7.1 ± 1 80 ± 10 35

6B 7.1 ± 1 80 ± 10 35

7F 7.1 ± 1 80 ± 10 35

9V 7.1 ± 1 80 ± 10 35

14 7.1 ± 1 80 ± 10 35

18C 7.1 ± 1 80 ± 10 35

19A 7.1 ± 1 80 ± 10 35

19F 7.1 ± 1 80 ± 10 35

22F 7.1 ± 1 80 ± 10 35 23F 7.1 ± 1 80 ± 10 35

33 7.1 ± 1 80 ± 10 35

Table 2

ADVANTAGES OF THE PRESENT INVENTION

[0057] The present disclosure provides a novel culture medium that is easy to produce and highly economic.

[0058] The present disclosure provides a culture medium that includes defined ingredients and components which do not adversely affect the cell surface chemistry of the bacteria. [0059] The present disclosure provides a culture medium that is devoid of animal components and thereby contamination of the same with reactive blood group substances or adventitious agents are reduced or eliminated.

[0060] The present disclosure provides a culture medium that includes defined ingredients and components and hence downstream purification of capsular pneumococcal polysaccharide derived from Streptococcus pneumoniae is made easy.

[0061] The present disclosure provides a culture mediumthat obviates the disadvantages associated with the known art.

[0062] The present disclosure provides a method for producing pneumococcal capsular polysaccharide that is technically and commercially feasible.

Claims

We Claim:

1. A culture medium for growth of serotypes of Streptococcus pneumoniae comprising KH₂PO₄, NaHC0₃, soya peptone, yeast extract, MgS0₄, NaCl, CaCl₂, glucose and L-cysteine or its salts.

2. The culture medium of claim 1 , wherein said glucose is D-glucose.

3. The culture medium of claim 1, wherein said MgS0₄ is MgS0₄7H₂0.

4. The culture medium of claim 1 , wherein said L-cysteine is hydrochloride salt of L-cysteine.

5. The culture medium of claim 1, comprising, on a per liter basis, 0.1 to 2g of KH₂P0₄, 0.1 to 2g of NaHC0₃, 1 to 50g of soya peptone, 1 to 30g of yeast extract, 0.01 to 2g of MgS0₄, 0.1 to 5g of NaCl, 0.01 to 2g of CaCl₂, 1 to 50g of glucose and 0.01 to 2g of L-cysteine.

6. The culture medium of any of the preceding claims, comprising, on a per liter basis, 0.5g of KH₂P0₄, 0.22g of NaHC0₃, 30g of soya peptone, 20g of yeast extract, 0.4g of MgS0₄.7H₂0, 2.5g of NaCl, O.Olg of CaCl₂, lOg of D-glucose and 0.15g of L-cysteine.HCl.

7. The culture medium of claim 1, wherein said serotype of Streptococcus pneumoniae is selected from the group consisting of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

8. A method for producing capsular pneumococcal polysaccharide, comprising the steps of:

(a) providing a culture medium comprising KH₂P0₄, NaHC0₃, soya peptone, yeast extract, MgS0₄, NaCl, CaCl₂, glucose and L-cysteine or its salts;

(b) growmgfitreptococcus pneumoniae in said culture medium;

(c) inactivating cells in culture broth;

(d) recovering capsular pneumococcal polysaccharide from said culture medium; and

(e) optionally purifying capsular pneumococcal polysaccharide obtained in step (d).

9. The method of claim 8, further comprising feeding said culture medium with glucose solution in step (b).