FIELD OF INVENTION
The present invention in general relates to the fields of microbiology and cell culture. The invention relates to improvement of culture and fermentation conditions for propagation and production of capsular polysaccharides from Streptococcus pneumoniae. The invention in particular relates to a novel culture medium free of serum and animal component for production of polysaccharide from S. pneumoniae. The polysaccharides obtained by the novel process may be further used for preparation of immunogenic compositions for preventing and treating infections caused by the organism.
BACKGROUND OF THE INVENTION
Streptococcus pneumoniae
Streptococcus pneumoniae, or pneumococcus, is gram-positive, lancet-shaped cocci, alpha-hemolytic, bile soluble aerotolerant anaerobe and a member of the genus Streptococcus. Streptococcus pneumoniae is a normal inhabitant of the human upper respiratory tract. It 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 pneumococcus, referring to its morphology and its consistent involvement in pneumococcal pneumonia.
Streptococcus pneumoniae is a fastidious bacterium, growing best in 5% carbon dioxide and complex medium. Nearly 20% of fresh clinical isolates require fully anaerobic conditions. In almost all cases, growth requires a source of catalase (e.g. blood) to neutralize the large amount of hydrogen peroxide produced by the bacteria. In complex media containing blood, at 37°C, the bacterium has a doubling time of 20-30 minutes (Todar's online textbook of bacteriology- http://www.textbookofbacterioloRy.net)
The ability of the pneumococcus to resist the major mechanism of clearance of the organism from the bloodstream (i.e. opsonophagocytosis) requires expression of the major virulence factor of the organism, which is a polysaccharide capsule. 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. Ninety different pneumococci serotypes have been identified and each serotype corresponds to a different chemical composition of the capsule. The capsule is currently used as an antigen in pneumococcal vaccines. Pneumococcal capsular polysaccharide vaccines have been licensed since 1977. The 23-valent unconjugated vaccine (Merck' PNEUMOVAX® 23) is designed to provide coverage against -90% of the most frequently reported isolates. This vaccine does not, however induces immune memory, hence is not effective in children below 2 years of age. Advances in the conjugation technology has led to conjugation of the polysaccharides to carrier proteins leading to a T-cell response, enabling its use in younger children (Wyeth's 7-valent Prevnar® and 13-valent Prevnar 13®, GSK's Synflorix®)
Culture media
Manufacturing of pneumococcal polysaccharide requires fermentation of S. pneumonia bacteria. Current good manufacturing practices (cGMP) are stringent on quality and selection of several criteria in medium development for microbial fermentation for the production of biologics, especially vaccines. Under cGMP fermentation procedures, quality is built into the entire process ensuring that the requirement of the regulatory agencies are met in terms of safety, product identity, quality and purity (FDA Title 21, Code of Federal Regulations, Parts 210, 211, and 600-680). Ideally, the medium should contain only essential components and should be easily prepared in a reproducible manner. A chemically defined medium is inherently more reproducible than a complex medium. Furthermore, a chemically defined medium enables discrete analysis of the effect of each component and strict control of medium formulation through identity and purity testing of raw materials. Finally, the fermentation medium should support the cultivation of the microorganism in question to high-cell density to improve volumetric productivity and to generate a final culture whose composition and physiological condition is suitable for downstream processing.
Fermentation, as the first unit operation in the process, has a major impact on other subsequent unit operations. Fermentation medium is an important parameter that can affect the entire process. Selection of the medium impacts the growth rate, productivity, cell viability, foaming, and by-product formation.

Media development hence is a vital part of GMP manufacturing and must meet the expectations of developing a scalable, cost-effective and robust process.
Various cell culture media for S. pneumoniae have been documented in the literature and a number of media are available commercially. Typically, most of the media used for the growth of fastidious organisms such as S. pneumoniae contain whole blood, (chocolate blood agar, charcoal medium), blood components such as Hemin (Robertson's cooked meat broth), egg yolk (Dorset Egg Media) or animal extracts. These components make the basal media nutritionally enriched, so as to support the growth of the fastidious bacteria.
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.
Furthermore, as such media are not chemically defined; there may be lot to lot variation in the composition of the medium. The use of undefined components such as serum and animal extracts also hinders the elucidation and definition of the appropriate and the critical nutritional requirements of the bacteria. The presence of these components in the medium further leads to increase in cost of purification due to the necessity of removing these process associated proteins, which if present may lead to adverse immunological reactions.
Few animal component free culture media for S. pneumonia are also known in the prior art. For example:
a) Appl Microbiol Biotechnol (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 Ind Microbiol Biotechnol (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, which is the focus of the present invention.
It is well known that the capsular polysaccharide of S. pneumoniae is the major virulence factor of the organism and provides an attractive immunogen for developing a vaccine against the pathogen. The media used for the production of the polysaccharide from S. pneumoniae in an economic mode should fulfill the following criteria:
• It should be free of serum components
• It should be free of animal components
• It should not pose difficulties in downstream processing
• Provide large scale production of polysaccharide in high yields and purity.
• It should be cost effective and exclude cost intensive components such as HEPES, Hemin, etc.
Thus, there lies a challenge in developing a medium, which is safe, serum and animal component free, reduces complexities and cost in downstream processing, is cost effective due to inclusion of cheaper chemicals and compounds and allows for large scale production of polysaccharide in high purity and yields, leading to preparation of immunogenic composition against the infection caused by the organism. The currently known and available media for cultivation and propagation of S. pneumoniae are mainly enriched media with blood and/ or animal components. This is not desirable as indicated above due to various disadvantages. Certain defined media without blood and animal products are in use, but the exclusion of the blood and/ or animal components leads to supplementing the media with number of other amino acids, vitamins etc. making the media expensive. Thus, there is a need for a medium for the growth and propagation of S. pneumoniae, which satisfies all of the above mentioned criteria.
The present invention relates to novel fermentation conditions and specifically a novel culture medium for S. pneumoniae. Such a medium would also have the advantage of not having large quantities of proteins and hence would not adversely modify the cell surface chemistry, which is very critical for the in vitro microenvironment of S. pneumoniae. Further, being a

defined medium, it would also aid in controlling the nutritional requirements of the bacteria for optimizing the fermentation process for specified application.
SUMMARY OF THE INVENTION
The present invention is based on the development of improved fermentation processes and conditions, which allow the cultivation of Streptococcus pneumoniae and production of capsular polysaccharide.
In the first aspect, the present invention provides a novel serum and animal component free Streptococcus pneumoniae fermentation media.
A particularly preferred medium of the invention is the one that is fully defined. Such a medium does not contain any components which are undefined, that is, the components whose content is unknown or which may contain undefined or varying factors that are unspecified.
In specific embodiments of the methods of the present invention described herein, the
cultivation medium comprises of Glucose, Soya peptone, Yeast extract, L-glutamine, L-
asparagine, Thioglycolic acid, Choline chloride, K2HPO4, NaHCO3, FeSO4, MgSO4, MnSO4
and ZnSO4.
The present invention meets the need of the art of providing media for growth and propagation of Streptococcus pneumoniae that is free of serum and animal components. The media of the invention eliminates the risk associated with the contamination occurring due to presence of the serum and animal components in the medium and provides a growth medium that affords the ease of downstream processing due to the absence of animal component related impurities. Further, it provides a medium that is cost effective by the virtue of the chemicals and components making up the medium composition. The media of the invention allows for good quantity of polysaccharide production by Streptococcus pneumoniae, which is the virulence factor of the organism and which may be further used for production of immunogenic composition against the infection by the bacteria.

BRIEF DESCRIPTION OF THE FIGURE
Figure 1 gives the measure of the biomass produced during the fermentation of various serotypes of S. pneumoniae.
Fermentation of each of the serotypes of S. pneumoniae was carried out as per the invention. The growth medium, feed medium and the growth parameters were adjusted according to the invention. At specified time intervals, a small aliquot of the ferment was taken for measurement of the optical density. The values obtained were plotted and illustrated graphically. The plot in the figure 1 gives the measurement of the optical densities, according to one aspect of the invention.
DESCRIPTION OF THE INVENTION
The present invention will be described with reference to certain embodiments but the present invention is not limited thereto but only by the claims.
The composition of media used to culture Streptococcus pneumoniae, is of paramount importance because of its influence on cell growth and production of essential antigenic components like polysaccharides. Conventional media comprise basal nutrient media supplemented with components like hemin, whole blood, serum, or other tissue fluids as these additives were thought to be required for growth. However, the use such conventional media is undesirable for several reasons. Growth media containing such components may vary in composition and contaminants, thereby introducing extraneous factors and/or infectious agents into the system. Recent concerns by the FDA, EMEA and others about animal component and /or serum component quality, contamination and increased demand have generated significant interest in the development and utility of serum-free and animal component free growth media.
Serum-free and animal component free media provide many important advantages, including lot-to-lot consistency, biological uniformity, and freedom from adventitious agents.
The present invention provides serum and animal component free media, for the growth and propagation of Streptococcus pneumoniae. These media can be seen as a solution to the problems of the prior art media for growth of S. pneumoniae, which were either serum and animal component based media or defined media comprising a list of amino acids, vitamins, etc., making the media cost intensive. Further, it provides a general composition for culturing

of different serotypes of S. pneumoniae with minor changes for the production of capsular polysaccharides.
Due to the exclusion of the serum and the animal components in the media of the invention, the risk of contamination associated with these components is eliminated. Also the exclusion of such components reduces the cost of the media. The cost is further reduced by exclusion of a long list of various amino acids and vitamins in the media. Due to the lower protein content of the media, there is an ease of downstream processing, which also further results in reduced processing and hence the product cost. Since the media comprises lower amounts of proteins, such a media also allows controlling the nutritional requirements for the growth and propagation of the organism and for optimal production capacity of the desired component. Such a media, by virtue of its low protein content, also does not adversely affect the microenvironment of the cells, in vitro, which is one of the major factors affecting the growth and production capacity of the anaerobes such as S. pneumoniae.
The media of the invention, in spite of its lower protein content allows for large scale production of polysaccharide by the S. pneumoniae, which is one of the virulence factors of the organism and which can further be used for production of immunogenic compositions against the infections caused by the bacteria.
Specifically, the present invention provides a chemically defined serum and animal component free medium comprising components selected from Glucose, Soya peptone, Yeast extract, L-glutamine, L-asparagine, Thioglycolic acid, Choline chloride, K2HPO4, NaHCO3, FeSO4, MgSO4, MnSO4 and ZnSO4.
Definitions
Anaerobe- Anaerobe is an organism that does not require oxygen for growth. Anaerobes may be obligate anaerobes, which could possibly react negatively and may even die in the presence of oxygen. The other types are the facultative anaerobes that make ATP by aerobic respiration if oxygen is present else grow in its absence also. Streptococcus pneumoniae is a facultative anaerobe.
Animal component free medium- Animal component free medium is a culture medium devoid of any animal derived material in its composition. Specifically such medium does not

contain any component, which has been purified from animals, particularly from animal serum and the like. Instead, such media use components, which are not directly obtained from animals.
Batch fermentation- Batch fermentation is one of the methods used in the industrial production of microorganisms, where the sterile growth medium is inoculated with the microorganisms and no additional growth medium is added.
Continuous fermentation- Continuous fermentation is a technique used to grow the microorganisms or cells continuously while maintaining a particular phase of growth. This type of fermentation is used when a culture is capable of producing the required product only in a particular growth phase such as the log phase.
Culture medium- Culture medium is a liquid or gel designed to support the growth of the microorganisms or cells. Such a medium may be customized for meeting the specific requirements of growth of the organism and/ or the purpose of its growth.
Culture used: The present invention is directed to growth and production of polysaccharides from S. pneumoniae. Preferably the serotypes selected are one or more, or all of 1,2, 3, 4, 5, 6B, 6A, 7F, 8, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33 F. The strain used may be a wild type or genetically modified strain.
Defined medium- A defined medium is a medium whose exact chemical composition is quantitatively known. Such type of a medium does not contain hydrolysates or components having an unknown composition.
Enriched medium- A media having a nutrient constituent, which encourages the growth of particular bacteria or microbe, such as the fastidious organisms. Fortified media are called enriched media, such as blood agar and chocolate agar.
Enhanced biomass production- Enhanced biomass production refers to the biomass achieved, measured in any known way, by the serotypes of S. pneumoniae, after the fermentation in the optimized medium, using optimal feed medium and using the process for fermentation along with the optimized growth parameters, according to the invention. The enhanced biomass may be measured by any method known in the art, such as dry weight,

wet weight, cell count, measuring colony forming units (CFUs), optical density measured at appropriate wavelength.
The enhanced biomass may be measured at the time the culture goes into log phase, or end of log phase or any other specified time.
Enhanced polysaccharide production- Enhanced polysaccharide production refers to the polysaccharide produced by the serotypes of S. pneumoniae, after the fermentation in the optimized medium, using optimal feed medium and using the process for fermentation along with the optimized growth parameters, according to the invention.
The yield of the polysaccharide may be measured before, after partial or final purification. The yield of polysaccharide so produced may be assayed and/ or calculated by any technique known in the art.
Fastidious organism- Fastidious organism is a bacterial organism having complex nutritional requirements.
Fed- batch fermentation- Fed- batch fermentation is one of the methods used in the industrial production of microorganisms wherein, the growth limiting substrate is continuously added to the fermentation vessel. This growth limiting substrate can be anything including carbon or nitrogen source, vitamins, essential amino acids, etc.
Fed- batch can be a fixed volume or a variable volume fed batch. In the fixed volume fed-batch the volume of the fermentation medium is kept almost the same by either supplying the feed in an undiluted form or in form of a gas. Another method of keeping the volume same is by supplying the feed by dialysis. Cyclic fed-batch culture for fixed volume systems refers to a periodic withdrawal of a portion of the culture and use of the residual culture as the starting point for a further fed-batch process. Basically, once the fermentation reaches a certain stage, the culture is removed and the biomass is diluted to the original volume with sterile water or medium containing the feed substrate. The dilution decreases the biomass concentration and result in an increase in the specific growth rate.
Variable volume fed-batch is one in which the volume changes with the fermentation time due to the substrate feed. The feed is provided in the same batch fermentation vessel so as to increase the initial volume of the medium. The feed may be the growth limiting substrate fed

at a concentration equal to its concentration in the initial medium or in concentrated form.
Immunogenic composition- An immunogenic composition is a formulation capable of provoking immune response when administered to a subject.
Serum free medium- Serum free medium is a medium free of serum proteins but including the minimal essential substances required for cell growth. This type of medium avoids the presence of extraneous substances that may affect cell proliferation or unwanted activation of cells.
Solution D- The composition of the solution D for the purpose of the invention is a solution containing 0.1-2 g/1 of L-glutamine, 0.1- 5 g/1 of L-asparagine and 0.01-1 g/1 of Choline Chloride and more preferably is a solution containing 0.62 g/1 of L-glutamine, 1 g/1 of L-asparagine and 0.1 g/1 of Choline Chloride.
Solution E- The composition of the solution E for the purpose of the invention is a solution containing 0.05- 2 g/1 of FeSO4. 7H2O, 0.1-10 ml of Thioglycolic acid, 0.01- 0.5 g ZnSO4. 7H2O and 0.005- 0.2 g of MnSO4.H2O and more preferably is a solution containing 0.5g/l of FeSO4. 7H2O, 1 ml of Thioglycolic acid, 0.08 g ZnSO4. 7H2O and 0.036 g of MnSO4. H2O
The media of the invention
The present invention is directed towards a medium for the growth and propagation of Streptococcus pneumoniae. The said medium is serum free as it does not have any of the serum derived proteins and / or is free of animal components.
According to one aspect of the invention, the medium has low protein content than a medium comprising serum or animal components.
According to a preferred aspect of the invention, the medium of the invention allows for improved growth of Streptococcus pneumoniae.
According to another preferred aspect of the invention, the medium of the invention allows for high polysaccharide production by the organism.

According to a yet another preferred aspect of the invention, the medium of the invention does not comprise of many amino acids and vitamins for supporting the growth and polysaccharide production by Streptococcus pneumoniae.
Another aspect of the invention relates to a medium that comprises defined ingredients and components, such that the cost of production of media is low.
Further aspect of the invention relates to a media comprising defined ingredients and components such that the downstream purification of the product derived from S. pneumoniae grown in the said medium, is cost effective and can be performed with relative ease.
One further aspect of the invention affords a medium that comprises defined ingredients and components, which do not adversely affect the cell surface chemistry of the bacteria, which is very important for certain cell types while being grown in vitro, for the maintenance of the cellular microenvironment.
Components of the media of the invention
The formulation of the medium of the invention is such that it does not comprise serum or animal derived components in its composition. However, such a medium has all the ingredients necessary for supporting and improving the growth of Streptococcus pneumoniae and allowing improved production of capsular polysaccharide, which is one of the major virulence factors of the bacteria.
According to one aspect of the invention, the components of the media are such that they afford ease and cost effectiveness to the downstream processing and thus reduce the final production cost and efforts.
According to one preferred aspect of the invention, the components of the medium of the invention are such that they do not add up to such a high protein content that they adversely affect the microenvironment of the bacteria grown in vitro.
According to a specific aspect of the invention, the medium of the invention comprises one or more of carbon sources selected from a group comprising of glucose, fructose, sucrose, xylose, mannitol, sorbitol, lactose, galactose, and the like.

According to a preferred aspect of the invention, the medium of the invention comprises of glucose as the preferred carbon source.
According to yet another specific aspect of the invention, the medium of the invention comprises one or more nitrogen sources selected from a group comprising of amino acids, ammonium salts, nitrates, nitrites, peptones, yeast extract, casamino acids, and the like.
According to a preferred aspect of the invention, soya peptone and yeast extract are preferred, as nitrogen sources.
According to another preferred aspect of the invention, the medium of the invention
comprises,
L- Glutamine and L- Asparagine.
According to another aspect of the invention, the medium of the invention may comprise one
or more or all of sulfur, phosphorus, potassium, magnesium, calcium, oxygen sources,
various trace elements and growth factors.
MgSO4, preferably MgSO4.7H2O is the source of magnesium present in the medium of the invention, according to a preferred aspect of the invention.
One further aspect of the invention refers to the use of one or more of sulfur containing amino acids, thioglycolic acid, calcium thioglycolate and sodium thioglycolate, sulfur thioglycolate, sulphates, sulfites, etc. as the sulfur source in the medium of the invention.
One preferred aspect of the invention refers to the use of thioglycolic acid in the medium of the invention as a source of sulfur.
Another aspect of the invention deals with the inclusion of one or more of nucleic acids and phospholipids in the medium of the invention, as a source of phosphorous.
According to one aspect of the invention, one or more of copper, iron, molybdenum, cobalt, zinc, manganese, etc. may be present in the medium of the invention as trace elements. FeSO4, preferably FeSO4. 7H2O, ZnSO4, preferably, ZnSO4. 7H2O and MnSO4, preferably

MnSO4. H20, are the trace elements present in the medium of the invention, as per one preferred aspect.
According to one further aspect of the invention, one or more of amino acids and/ or vitamins may be present in the medium of the invention as growth factors.
According to one preferred aspect of the invention, the medium of the invention comprises Choline chloride.
According to one aspect of the invention, the medium of the invention comprises one or more of buffers such as HC1- NaOH buffer, phosphate buffer, citrate buffer, carbonate buffer, HEPES buffer, etc. or any other salts for the maintenance of the ionic strength of the medium.
According one preferred aspect of the invention, the medium of the invention comprises one or both of K2HPO4, NaHCO3.
In a most preferred aspect of the invention the medium of the invention comprises Glucose, Soya peptone, Yeast extract, L-glutamine, L-asparagine, Thioglycolic acid, Choline chloride, K2HPO4, NaHCO3,FeSO4.7H2O, MgSO4.7H2O, MnSO4.H2O and ZnSO4.7H2O.
In another preferred aspect of the invention the medium of the invention has the following specific composition.
(Table Removed)
According to the most preferred aspect of the invention, the composition of the medium of the invention is as under:
(Table Removed)
Physiological parameters affecting the growth of S. pneumoniae
Apart from the nutrient requirements, each organism has a specific set of physiological needs. The optimum growth of the organism during fermentation is dependent on these physiological parameters. These parameters include, temperature of growth, pH, oxygen content (dissolved O2 in the medium), agitation, ionic strength, etc. of the medium.
Under certain conditions, an organism may be able to grow to give higher biomass than other set of conditions. Similarly, the production capacity may change depending upon the type of parameters applied for the growth of the organism. Variations in temperature, pH, O2 content, etc. may lead to changes in the metabolic pathways, which may in turn change the way a substrate is utilized, or a product is produced. Even slightest change may lead to significant variations.
According to a preferred aspect of the invention the pH of the medium of the invention is in range of 6- 8.
According to a more preferred aspect of the invention, the pH of the medium of the invention
is between 6.8-7.3.
The temperature of the fermentation for the purpose of the invention is preferably optimal for the growth of the organism, more preferably between 30- 45°C and most preferably between 32-40°C.
According to a preferred embodiment of the invention, the agitation used for the fermentation is 10- 200 rpm and more preferably 50-110 rpm.
There may not be a single set of the growth or process parameters that may lead to the optimal growth or saccharide production by each of the serotypes of S. pneumoniae. The optimization of these physiological parameters may be required for each of these serotypes, to attain optimal growth and saccharide production. This has to be done in lab by employing novel permutation and combination of various parameters.
One way of doing this is by keeping all the parameters to be studies constant except for one, the effect of which may be recorded on the end points; the endpoints being biomass production and the saccharide production by the organism, in this case. When the value of the said parameter is obtained which seems to be the optimal one, this parameter may then be kept constant along with the others under study. In this case some other parameter may be varied in similar way to obtain the optimal value of the same. Once the optimal values of each of the growth parameters is obtained, the fermentation may be carried out under the arrived optimal conditions to find out the difference in the yields obtained before and after the optimization. Further fine tuning of the process parameters may be done if required. A similar or any other strategy may be used for each of the process parameters that need to be optimized and this has to be done separately for each of the serotypes of S. pneumoniae.
One embodiment of the invention deals with the use of same or similar physiological or process parameters for the fermentation of various serotypes of S. pneumoniae.
According to another embodiment of the invention, one or more physiological or process parameters may vary depending upon the type of the serotype of S. pneumoniae being used for fermentation.
One another embodiment of the invention, deals with the use of one or more different process
parameters for the fermentation of same type of S. pneumoniae serotype.
According to one preferred embodiment of the invention, the various physiological or process parameters used for the fermentation of different serotypes of S. pneumoniae are as given in the table below:
(Table Removed)
Fermentation conditions
The inoculum preparation for the seeding of the bioreactor for the purpose of the fermentation, according to one embodiment of the invention, may be done by known standard techniques. The culture may be inoculated from the frozen stock, into small flasks, containing the culture medium. The culture may be grown under appropriate conditions until the optimum optical density is achieved. The inoculum may be checked for identity and purity and may be used for the seeding of the bioreactor for fermentation.
According to one preferred embodiment of the invention, the medium used for the fermentation is same as that of the inoculum medium.
The fermentation method used may be batch, fed- batch or continuous fermentation. The type of the fermentation employed would be dependent on the type of organism, the type of product and the growth phase in which the product is produced by the organism.
According to one aspect of the invention, the fermentation employed is dependent on the organism, S. pneumoniae and the growth phase and the other conditions, which are conducive for better biomass production.
According to one preferred aspect of the invention, the fermentation employed is dependent on the organism, S. pneumoniae and the growth phase and the other parameters conducive for better polysaccharide production by the organism.
According to another aspect of the invention, the fermentation employed for biomass production by S. pneumoniae is either batch, fed- batch or continuous fermentation.
According to one preferred aspect of the invention, the fermentation employed for polysaccharide production by S. pneumoniae is either batch, fed- batch or continuous fermentation.
One more preferred aspect of the invention provides that the type of fermentation employed for polysaccharide production by S. pneumoniae is fed- batch fermentation.
The invention specifically provides an improved method of culturing S. pneumoniae using a fed-batch process on a manufacturing scale. Fed-batch culture may be either fixed volume fed-batch or variable volume fed-batch. In fixed volume fed batch culture, the limiting substrate is fed without diluting the culture (e.g., using a concentrated liquid or gas or by using dialysis). In variable volume fed batch culture, the volume changes over fermentation time due to the substrate feed.
To improve polysaccharide production, fed-batch fermentation has been examined using different feed solutions and feeding strategies.
According to an embodiment of the invention, the bioreactor may be inoculated with a specific volume of the inoculum, at a specific temperature and rate of agitation (rpm), which is suitable for the fermentation of the culture. Samples may be drawn at specific intervals, preferably at each hour, for monitoring the growth, purity and integrity of the culture. The feed may be initiated at a specific rate, when the culture in the bioreactor reaches a specific optical density.
According to one preferred embodiment of the invention, the composition of the feed to be used during the fed-batch may have the following composition:
(Table Removed)
According to a more preferred embodiment of the invention, the composition of the feed to be used during the fed- batch may have the following composition:
(Table Removed)
The requirement of the various media components for various types of organisms may vary. Each organism would grow optimally only when an optimal combination of the various growth nutrients are provided by the growth medium. These requirements for each of the organism may also be different for optimal production of certain proteins, saccharides, etc. These requirements may also vary from one serotype of S. pneumoniae to other.
The optimization of the concentration and amounts of these growth nutrients have to be done in lab to arrive at a perfect combination to allow optimal biomass and saccharide production
by each of the serotypes of S. pneumoniae. This has to be done by performing various lab scale experiments employing various permutation and combinations of the various ingredients of the growth media.
Similarly, it is required that the feed medium used for the feeding of the culture during fermentation, in a fed- batch, be optimized for allowing optimal biomass and saccharide production. The optimization of the feed has to be done keeping in mind that an organism may produce the desired substance only when a particular nutrient is limiting. In other cases, it may be required that for the optimal production of a desired substance, a particular nutrient should not be anytime in limitation. In absence of such an optimal feed, the fermentation may not proceed in a desired manner leading to low biomass or low saccharide production, in case of S. pneumoniae. Thus, this optimization is very critical and requires a lot of research in the wet lab, as a small tilt to the other side may result in entire different results, than that desired. According to one embodiment of the invention, the amounts and the concentrations of the various components of the media used are same for the fermentation of all the serotypes.
Another embodiment of the invention deals with use of different amounts and/ or different concentrations of one or more components of the media for the fermentation of different serotypes of S. pneumoniae.
One more embodiment of the invention refers to the use of media having different amounts
and/ or concentrations of the components, for the fermentation of same type of serotype of S.
pneumoniae.
According to one preferred embodiment of the invention, the composition of the media used
for the fermentation of various serotypes of S. pneumoniae, according to the invention, may
be depicted as in the following table.
(Table Removed)
According to a more preferred embodiment of the invention, the above table refers to the composition of the feed medium used for the fermentation of the various serotypes of S. pneumoniae, as referred to in the above table.
The pH of the medium for fermentation may be maintained using HCl and/ or NaOH or any other suitable buffer system.
According to one embodiment of the invention, antifoaming agents such as silicones, glycerol, alcohols, organic phosphates, glycols, etc., may be added to the fermentation media.
When the culture reaches stationary phase, the run may be continued for some time as determined earlier, before the termination of the fermentation.
On termination of the batch, the culture may be harvested and the cells may be separated from the spent medium by standard techniques known in the art.
According to one aspect of the invention, the novel growth medium, optimized growth parameters employed during the cultivation and the propagation of the bacteria, the optimized feed medium employed, along with the other factors, result in a better biomass production during fermentation.
The biomass produced during the growth, cultivation or fermentation of the bacteria, may be measured by any of the known means in the prior art. The measurement may be done by taking the dry or wet cell weight, by cell counting, by counting the colony forming units
(CFUs), by measuring optical density (OD) at appropriate wavelength.
According to one preferred aspect of the invention, the biomass produced during the
fermentation, is measured by measuring OD at any appropriate wavelength.
One more preferred aspect of the invention deals with the measurement of the biomass done by measuring the OD at 600 nm.
During the cultivation, growth or fermentation, aliquots of the culture may be taken and used for the measurement of the biomass. The aliquots removed may be centrifuged to take the wet weight of the pellet obtained, or dried by various techniques known in the art, to monitor the dry weight of the pellet. The aliquots may also be used for cell counting or used for inoculating growth medium for determining the colony forming units (CFUs).
According one preferred aspect of the invention, the aliquots drawn are used to determine optical density of the culture. The determination is done at specified intervals and the values are plotted for graphical illustration.
According to another preferred aspect of the invention, the optimized growth medium, the optimized feed medium and the process used for fermentation of the bacteria, along with the optimal growth parameters, according to the invention, resulted in enhanced biomass for each of the serotype at specific time.
The inactivation of the culture may be done before or after the harvesting and separation of the cells from the medium. The inactivation of the culture may be affected by any known chemical, physical or enzymatic means known in the art.
One preferred embodiment of the invention provides that the inactivation of the culture may be done using formalin at specific concentration and a sample may be tested for checking complete inactivation of the cells.
Release of the polysaccharide from the cell surface may be done before or after the inactivation of the cells. According to a preferred embodiment of the invention, the polysaccharide may be released from the surface of the cells, post-inactivation. The release of the polysaccharide from the cell surface may be affected by known physical, mechanical or
enzymatic treatment. According to a more preferred embodiment of the invention, the inactivated cells may be subjected to treatment with sodium deoxycholate (DOC) at a desired concentration for release of the polysaccharide from the surface of the cell. The polysaccharide prepared according to this invention may be separated from the cells and purified by standard techniques known in the art. In one embodiment of the invention, the broth may be filtered to separate the cells from the spent medium. The polysaccharide may be separated from other sub cellular fractions by various methods, using detergents, pH changes and alcohol. The debris may be removed to get a very crude preparation of the polysaccharide. The purification of the crude sample may be affected by use of methods such as dialysis and precipitation of the polysaccharide using alcohol, cationic detergents etc. Further purification may be affected by using methods such as centrifugation, precipitation, ultra-filtration and column chromatography.
The polysaccharide so obtained may be assessed for purity and further characterization on the basis of molecular weight, sugar content, spectroscopic analyses, etc. The cultivation, propagation and the fermentation of the bacteria need to be done in a fashion so as to obtain not only good biomass yields but also better saccharide production, since finally the saccharide is the antigen that is to be extracted to be used in production of immunogenic composition against the infection by S. pneumoniae. Not all medium and the type of parameters used for the fermentation of the bacteria, allow both, good biomass production as well as good saccharide yields. S. pneumoniae serotypes are known to produce more biomass under a specific set of conditions including the medium and the growth parameters and give better saccharide yields in other set of growth conditions. However, a lot of experimentation is required to attain specific growth and cultivation conditions to allow the bacteria to grow to attain appreciable biomass along with better saccharide yields, separately for each of the serotypes.
The invention provides that the novel process involving the use of the novel medium for cultivation, the optimized growth parameters, the specific growth conditions, taken together with the optimized feed medium for the fed- batch and the subsequent conditions and parameters employed, lead to an enhanced biomass as well as increased polysaccharide production, by each of the serotypes.
The polysaccharide for the purpose of the invention may be prepared from one or more
strains of streptococcus, viz; 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 20F, 23F, 33F, or any other known pneumococcal strain. The strain used may be either wild type or genetically modified strain. The present invention presents a monovalent or multivalent immunogenic composition comprising the polysaccharides from various strains. The polysaccharides used for the preparation of the vaccine according to the present invention, may be prepared by standard known techniques. The size and the modification of the polysaccharides may be such that it provides good immune response against the respective pneumococcal strains. The polysaccharide may be used as such or in form of a protein- polysaccharide conjugate for preparation of immunogenic composition. These polysaccharides or conjugates may be formulated in a single dose formulation.
According to one preferred embodiment of the invention, the vaccine or the immunogenic composition of the invention may comprise one or more or all of the serotypes selected from the group of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.
The immunogenic composition according to one embodiment of the invention may comprise certain polysaccharides in un-conjugated form and certain polysaccharides in conjugated forms. For preparing the protein- polysaccharide conjugates, the purified polysaccharides may be chemically activated to make them capable of reacting with carrier proteins. Once activated, the polysaccharide may be conjugated to a carrier protein. The carrier proteins used are preferably the proteins that are non-toxic and non-reactogenic and obtainable in sufficient amount and purity. Carrier proteins should be amenable to standard conjugation procedures. The carrier protein used may be any of the carrier proteins known in the art, such as tetanus toxoid (TT), diphtheria toxoid (DT), CRM 197 and outer membrane protein of Neisseria meningitides and protein D from Haemophilus influenzae. Cholera toxoid (PCT application no. W02004/083251), E. coli LT, E. coil ST, exotoxin A from Pseudomonas aeruginosa, bacterial outer membrane proteins such as outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), C5a peptidase from Group A or Group B streptococcus, can also be used. Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD) can also be used as carrier proteins. In a multivalent formulation each polysaccharide may be conjugated to same or different carrier proteins, or may be left unconjugated.
The conjugation may be done by known standard techniques such as use of l-cyano-4-
dimethylamino pyridinium tetrafluoroborate (CDAP) for activation of the saccharide, reductive amination, etc. The linkage chemistry may be direct or indirect. The activated saccharide may thus be coupled directly or via a spacer (linker) group to an amino group on the carrier protein. The ratio of the protein to the polysaccharide in the conjugate may be between 1:10 - 10: 1. The immunogenic composition of the present invention may optionally also comprise free carrier proteins that may enhance the immunogenicity of the polysaccharides.
After conjugation of the capsular polysaccharide to the carrier protein, the polysaccharide-protein conjugates may be purified (enriched with respect to the amount of polysaccharide-protein conjugate) by a variety of techniques. After the purification of the protein-polysaccharide conjugates, they may be formulated into the immunogenic composition of the present invention, which can be used as a vaccine.
Compositions of the invention may comprise pharmaceutically acceptable ingredients and may be typically in aqueous or fully liquid form; the composition may also be available as a lyophilized powder, to be reconstituted. The composition of the invention may comprise non-active ingredients including adjuvant, buffer, isotonic agent, preservative, stabilizer, salt, etc.
The method for immunization according to the present invention will make use of a prophylactically effective amount of the pneumococcal polysaccharide. The immunogenic composition of the invention may be administered to children as well as adults for the treatment of the infections caused by Streptococcus pneumoniae. The vaccine formulations according to the present invention may be used to protect or treat a subject susceptible to pneumococcal infection, by means of administering the vaccine via a systemic or mucosal route.
The dosage regimen also may be deduced from studies involving immune responses of the subject, on administering the formulation of the invention. Following initial vaccination, the subjects may receive one or more booster doses appropriately spaced.
The vaccine of the invention may be available in form of kits. A kit or composition may be packaged (e.g. in the same box) with a leaflet including details of the vaccine e.g. instructions for administration, details of the antigens within the vaccine, storage instructions, etc. Embodiments herein relating to "vaccine" of the invention are also applicable to embodiments relating to
"immunogenic compositions" of the invention, and vice versa.
The following examples are used to further illustrate the present invention and advantages thereof. The following specific examples are given with the understanding that these are intended to be illustration without serving as a limitation on the scope of present invention.
Example I
This example gives the composition of the medium of the invention as per one of the
aspect of the invention -
Table 1
(Table Removed)
Example II
This example gives the procedure for carrying out the fermentation for production of
polysaccharide according to one of the aspect of the invention
For inoculum preparation for fermentation, Erlenmeyer flasks containing culture medium (at 1:5) ratio was inoculated with 0.03% (v/v) of the cell bank. The flask was incubated in CO2 incubator at 37°C and 5% CO2 for 7-8 hours and once the desired OD at 600nm of the culture was achieved the fermentor was inoculated with 5% (v/v) of the inoculum at temperature between 32- 40°C and agitation rate between 50-160 rpm. When the OD reached 0.8- 1.5 AU, feed was initiated at the rate of 1-1.3 ml/ min/ 1.
Composition of the feed according to one embodiment of the invention is:
Table 2
(Table Removed)
When the growth reached stationary phase air was passed at 0.1 wm for about 1 hour.
The culture was inactivated with 0.2- 0.25 % formalin at 25°C and the sample was checked for complete inactivation. After inactivation, sodium deoxycholate (DOC) was added to the final concentration of 0.1-0.2% (w/v) for the release of the polysaccharide from the surface. After DOC treatment, the culture was harvested in a sterilized bottle and centrifuged at 10,000g for 60 minutes at 5+/- 3°C. The pellet was discarded and the supernatant was taken for further purification.
Example III
This example gives the compositions of the feed media used for carrying out the
fermentation of various serotypes of S. pneumoniae, according to one of the aspect of the
invention
Table 3
(Table Removed)
Example IV
This example gives the process parameters used for carrying out the fermentation of
various serotypes of S. pneumoniae, according to one of the aspect of the invention
Table 4
(Table Removed)
Example V
This example gives the measure of the biomass produced during the fermentation of
various serotypes of S. pneumoniae, according to one aspect of the invention
The optimized growth medium, the optimized feed medium and the process used for fermentation of the bacteria, along with the optimal growth parameters, according to the invention, resulted in enhanced biomass for each of the serotype at specific time, as given in the table below:
Table 5
(Table Removed)
Example VI
This example gives the yields of polysaccharides on the partial purification of the broth obtained on carrying out the fermentation of various serotypes of S. pneumoniae, according to one of the aspect of the invention
Each of the fermented broth obtained, on the fermentation of various serotypes of S. pneumoniae, was subjected to partial purification. The purification was done according to the standard known techniques as already mentioned in the above text. The yields obtained for each of the serotypes are depicted as given in the table below:
Table 6
(Table Removed)

We claim:
1. A novel culture medium free of serum and animal component for production of biomass and polysaccharide from serotypes of S. pneumoniae.
2. The novel culture medium as claimed in claim 1, wherein the serotypes of S. pneumoniae are selected from the group of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.
3. The novel culture medium as claimed in claim 1, comprising Glucose, Soya Peptone, Yeast extract, K2HPO4, NaHCO3, L- Glutamine, L- Asparagine and MgS04, FeS04, ZnS04, MnS04, Thioglycolic acid and Choline or its salts.
4. The novel culture medium as claimed in claim 3, comprising Glucose, Soya Peptone, Yeast extract, K2HPO4, NaHCO3, L- Glutamine, L- Asparagine and MgSO4, FeSO4. 7H20, ZnSO4. 7H20, MnS04. H20, Thioglycolic acid and Choline Chloride.
5. The novel culture medium as claimed in claim 4, comprising 1-50 g/1 Glucose, 1-50 g/1 Soya Peptone, 1-50 g/1 Yeast extract, 0.1- 10 g/1 K2HPO4, 0.01-5 g/1 NaHCO3, 0.1-2 g/1 L- Glutamine, 0.1- 5 g/1 L- Asparagine and 0.01-0.2 g/1 MgS04, 0.05- 2 g/1 FeS04. 7H2O, 0.01- 0.5 g ZnSO4. 7H2O, 0.005- 0.2 g MnSO4. H2O, 0.1-10 ml Thioglycolic acid and 0.01-1 g/1 Choline Chloride.
6. The novel culture medium as claimed in the claim 5, comprising 20 g/1 Glucose, 20 g/1 Soya Peptone, 20 g/1 Yeast extract, 5 g/1 K2HPO4, 1 g/1 NaHCO3, 0.62 g/1 L-glutamine, 1 g/1 L- Asparagine and 0.05 g/1 MgSO4, 0.5g/l FeSO4. 7H2O, 0.08 g ZnSO4. 7H2O, 0.036 g MnS04. H2O, 0.1-10 ml Thioglycolic acid and 0.1 g/1 Choline Chloride.
7. The novel culture medium as claimed in the claim 1, comprising Glucose, Soya Peptone, Yeast extract, L- Glutamine, L- Asparagine and MgSO4, FeSO4, ZnSO4, MnSO4, Thioglycolic acid and Choline or its salts.
8. The novel culture medium as claimed in the claim 7, comprising Glucose, Soya Peptone, Yeast extract, L- Glutamine, L- Asparagine, MgSO4, FeSO4. 7H20, ZnSO4. 7H2O, MnSO4. H2O, Thioglycolic acid and Choline Chloride.
9. The novel culture medium as claimed in the claim 8, comprising 50-400 g/1 Glucose, 10-200 g/1 Soya Peptone, 10-200 g/1 Yeast extract, 0.1-2 g/1 L- Glutamine, 0.1- 5 g/1 L- Asparagine, 0.01-5 g/1 MgSO4, 0.05- 2 g/1 FeSO4. 7H20, 0.01- 0.5 g ZnSO4. 7H20, 0.005- 0.2 g MnSO4. H20, 0.1-10 ml Thioglycolic acid and 0.01-1 g/1 Choline Chloride.
10. The novel culture medium as claimed in the claim 9, comprising 100-300 g/1 Glucose, 40-150 g/1 Soya Peptone, 40-140 g/1 Yeast extract, 0.1-2 g/1 L- Glutamine, 0.1- 5 g/1 L- Asparagine, 0.25- 1.0 g/1 MgSO4, 0.05- 2 g/1 FeSO4. 7H20, 0.01- 0.5 g ZnSO4. 7H20, 0.005- 0.2 g MnSO4. H20, 0.1-10 ml Thioglycolic acid and 0.01-1 g/1 Choline Chloride.
11. A process of fermentation of S. pneumoniae cells comprising the steps of-

a) growing the bacteria in the medium free of serum and animal components, under optimal conditions,
b) optionally, inactivating the separated cells,
c) optionally, releasing the polysaccharide from the cell surface,,
d) optionally, separating the cells from the broth,
e) optionally purifying of the polysaccharide.
12. The process as claimed in claim 11, wherein the fermentation of the bacteria maybe
done by batch, fed- batch or continuous fermentation.
13. The process as claimed in claim 12, wherein the fermentation of the bacteria is done
using fed-batch fermentation.
14. The process as claimed in claim 11, wherein the medium as claimed in claims 7-10
is used as a feeding medium.
15. The process as claimed in claim 14, wherein the feeding medium for different S.
pneumoniae serotypes is as given in the following table:
(Table Removed)
16. The process as claimed in claim 11, wherein the optimal conditions employed for
the fermentation are- pH- 6- 8, temperature- 30- 45°C, agitation- 10- 200 rpm.
17. The process as claimed in claim 16, wherein the optimal conditions employed for
the fermentation of each of the S. pneumoniae serotype as given in the table below:
(Table Removed)
18. The process as claimed in claim 11, wherein the inactivation is done using 0.2- 0.25 % formalin at 25°C.
19. The process as claimed in claim 11, wherein 0.1-0.2% (w/v) sodium deoxycholate (DOC), is used for releasing the polysaccharide from the cell surface.
20. The process as claimed in claim 11, which results in enhanced biomass production as well as enhanced polysaccharide production, as given in the table below:
(Table Removed)