WO2011151841A1 - Procédé de fermentation pour streptococcus pneumoniae - Google Patents

Procédé de fermentation pour streptococcus pneumoniae Download PDF

Info

Publication number
WO2011151841A1
WO2011151841A1 PCT/IN2011/000365 IN2011000365W WO2011151841A1 WO 2011151841 A1 WO2011151841 A1 WO 2011151841A1 IN 2011000365 W IN2011000365 W IN 2011000365W WO 2011151841 A1 WO2011151841 A1 WO 2011151841A1
Authority
WO
WIPO (PCT)
Prior art keywords
medium
fermentation
pneumoniae
polysaccharide
culture medium
Prior art date
Application number
PCT/IN2011/000365
Other languages
English (en)
Inventor
Rajesh Jain
Kapil Maithal
Original Assignee
Panacea Biotec Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Panacea Biotec Limited filed Critical Panacea Biotec Limited
Publication of WO2011151841A1 publication Critical patent/WO2011151841A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • 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.
  • Streptococcus pneumoniae 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://ww-w.textbookofbacteriology.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.
  • 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 ® )
  • cGMP Current good manufacturing practices
  • cGMP Current good manufacturing practices
  • 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, 21 1 , and 600-680).
  • 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.
  • 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.
  • 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.
  • 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:
  • the present invention relates to novel fermentation conditions and specifically a novel culture medium for S. pneumoniae.
  • a novel culture medium for S. pneumoniae 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.
  • 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.
  • 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.
  • the cultivation medium comprises of Glucose, Soya peptone, Yeast extract, L-glutamine, L-asparagine, Thioglycolic acid, Choline chloride, K 2 HP0 4 , NaHC0 3 , FeS0 4 , MgS0 4 , MnS0 4 and ZnS0 4 .
  • 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.
  • Figure 1 gives the measure of the biomass produced during the fermentation of various serotypes of S. pneumoniae.
  • 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.
  • 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.
  • 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. 1 000365
  • 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, K 2 HP0 4; NaHC0 3 , FeS0 4 , MgS0 4 , MnS0 4 and ZnS0 4 .
  • 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.
  • 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.
  • 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.
  • 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.
  • CFUs colony forming units
  • 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. T/IN2011/000365
  • 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.
  • 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 FeS0 4 . 7H 2 0, 0.1-10 ml of Thioglycolic acid, 0.01- 0.5 g ZnS0 4 .
  • 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.
  • the medium has low protein content than a medium comprising serum or animal components.
  • the medium of the invention allows for improved growth of Streptococcus pneumoniae.
  • the medium of the invention allows for high polysaccharide production by the organism.
  • 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.
  • the formulation of the medium of the invention is such that it does not comprise serum or animal derived components in its composition.
  • 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.
  • 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.
  • 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.
  • 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.
  • the medium of the invention comprises of glucose as the preferred carbon source.
  • 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.
  • soya peptone and yeast extract are preferred, as nitrogen sources.
  • the medium of the invention comprises, L- Glutamine and L- Asparagine.
  • 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.
  • MgS0 4 preferably MgS0 4 .7H 2 0 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.
  • one or more of copper, iron, molybdenum, cobalt, zinc, manganese, etc. may be present in the medium of the invention as trace elements.
  • FeS04 preferably FeS04. 7H20, ZnS04, preferably, ZnS04. 7H20 and MnS04, preferably MnS04. H20, are the trace elements present in the medium of the invention, as per one preferred aspect.
  • one or more of amino acids and/ or vitamins may be present in the medium of the invention as growth factors.
  • the medium of the invention comprises Choline chloride.
  • 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.
  • the medium of the invention comprises one or both of K 2 HP04, NaHC0 3 .
  • the medium of the invention comprises Glucose, Soya peptone, Yeast extract, L-glutamine, L-asparagine, Thioglycolic acid, Choline chloride, K 2 HP0 4 , NaHC0 3, FeS0 4 .7H 2 0, MgS0 4 .7H 2 0, MnS0 4 .H 2 0 and ZnS0 4 .7H 2 0.
  • the medium of the invention has the following specific composition.
  • composition of the medium of the invention is as under: MEDIA AMOUNT/
  • 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 0 2 in the medium), agitation, ionic strength, etc. of the medium.
  • an organism may be able to grow to give higher biomass than other set of conditions.
  • the production capacity may change depending upon the type of parameters applied for the growth of the organism. Variations in temperature, pH, 0 2 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.
  • the pH of the medium of the invention is in range of 6- 8.
  • 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.
  • the agitation used for the fermentation is 10- 200 rpm and more preferably 50-110 rpm.
  • 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.
  • one or more physiological or process parameters may vary depending upon the type of the serotype of S. pneumoniae being used for fermentation. T/IN2011/000365
  • 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.
  • the various physiological or process parameters used for the fermentation of different serotypes of S. pneumoniae are as given in the table below:
  • the inoculum preparation for the seeding of the bioreactor for the purpose of the fermentation 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.
  • 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.
  • 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.
  • 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.
  • the fermentation employed for biomass production by S. pneumoniae is either batch, fed- batch or continuous fermentation.
  • 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.
  • the limiting substrate is fed without diluting the culture (e.g., using a concentrated liquid or gas or by using dialysis).
  • variable volume fed batch culture the volume changes over fermentation time due to the substrate feed.
  • 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.
  • composition of the feed to be used during the fed-batch may have the following composition:
  • composition of the feed to be used during the fed- batch may have the following composition:
  • 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 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.
  • 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.
  • 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.
  • 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.
  • antifoaming agents such as silicones, glycerol, alcohols, organic phosphates, glycols, etc.
  • silicones such as silicones, glycerol, alcohols, organic phosphates, glycols, etc.
  • the run may be continued for some time as determined earlier, before the termination of the fermentation.
  • the culture may be harvested and the cells may be separated from the spent medium by standard techniques known in the art.
  • the novel growth medium optimized growth parameters employed during the cultivation and the propagation of the bacteria, the optimized 1 000365
  • 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.
  • 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.
  • 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).
  • CFUs colony forming units
  • 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.
  • 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.
  • 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.
  • 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.
  • DOC sodium deoxycholate
  • the polysaccharide prepared according to this invention may be separated from the cells and purified by standard techniques known in the art.
  • 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 0365
  • 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, 1 1A, 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.
  • 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.
  • 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 2011/000365
  • 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.
  • 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.
  • 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.
  • CDAP l-cyano-4- dimethylamino pyridinium tetrafluoroborate
  • 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.
  • 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.
  • kits 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.
  • kits 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.
  • This example gives the procedure for carrying out the fermentation for production of polysaccharide according to one of the aspect of the invention
  • 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
  • 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
  • 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:
  • 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

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention se rapporte à l'amélioration des conditions de culture et de fermentation pour la propagation et la production de biomasse et de polysaccharides capsulaires par Streptococcus pneumoniae. L'invention concerne en particulier un nouveau milieu de culture exempt de sérum et de constituant d'origine animale pour la production de biomasse et de polysaccharide. Les nouveaux milieux de croissance optimisés, les paramètres de croissance optimisés employés pendant la culture et la propagation des bactéries, associés aux autres facteurs, aboutissent à une meilleure production de biomasse ainsi qu'à de meilleurs rendements de saccharide par S. pneumoniae pendant la fermentation.
PCT/IN2011/000365 2010-05-31 2011-05-26 Procédé de fermentation pour streptococcus pneumoniae WO2011151841A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN1248/DEL/2010 2010-05-31
IN1248DE2010 2010-05-31

Publications (1)

Publication Number Publication Date
WO2011151841A1 true WO2011151841A1 (fr) 2011-12-08

Family

ID=44509516

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IN2011/000365 WO2011151841A1 (fr) 2010-05-31 2011-05-26 Procédé de fermentation pour streptococcus pneumoniae

Country Status (2)

Country Link
AR (1) AR082374A1 (fr)
WO (1) WO2011151841A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014038879A1 (fr) * 2012-09-07 2014-03-13 에스케이케미칼주식회사 Procédé de production d'un polysaccharide de capsule ayant un sérotype pneumococcique
WO2016207744A1 (fr) * 2015-06-26 2016-12-29 Biobridge Healthcare Solutions Pvt. Ltd. Procédé amélioré pour la production et la purification de polysaccharide pneumococcique capsulaire de streptococcus pneumoniae et procédé de production de composition de vaccin conjugué utilisant ledit polysaccharide
WO2017085602A1 (fr) * 2015-11-17 2017-05-26 Pfizer Inc. Milieux et procédés de fermentation pour la production de polysaccharides dans une culture de cellules bactériennes
CN107723337A (zh) * 2017-11-30 2018-02-23 成都欧林生物科技股份有限公司 筛选肺炎链球菌用固体培养基的制备方法
WO2021101904A1 (fr) * 2019-11-18 2021-05-27 Vaxcyte, Inc. Culture sans produit animal de bactéries streptococcus
IL265623B (en) * 2016-09-30 2022-12-01 Biological E Ltd Multivalent vaccines for lung spot that include polysaccharide-protein conjugates
CN117821342A (zh) * 2024-02-29 2024-04-05 北京民海生物科技有限公司 一种14型肺炎链球菌的发酵方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1982001995A1 (fr) * 1980-12-11 1982-06-24 Merieux Inst Procede de purification de polyosydes de streptococcus pneumoniae et vaccin a base de polyosides ainsi purifies
EP0071515A1 (fr) * 1981-07-30 1983-02-09 Laboratoires de Therapeutique Moderne L.T.M. Procédé d'obtention de polyosides capsulaires, polyosides capsulaires ainsi obtenus et leur application à la préparation de vaccins
WO1994009115A1 (fr) * 1992-10-15 1994-04-28 Merck & Co., Inc. PROCEDE DE CROISSANCE CLONALE DE $i(STREPTOCOCCUS PNEUMONIAE)
WO2004083251A2 (fr) 2003-03-17 2004-09-30 Wyeth Holdings Corporation Holotoxine du cholera mutante en tant qu'adjuvant et proteine de support d'antigene
WO2010080484A1 (fr) * 2008-12-18 2010-07-15 Wyeth Llc Procédé de contrôle du poids moléculaire d'un polysaccharide provenant de streptococcus pneumoniae en utilisant du carbone

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1982001995A1 (fr) * 1980-12-11 1982-06-24 Merieux Inst Procede de purification de polyosydes de streptococcus pneumoniae et vaccin a base de polyosides ainsi purifies
EP0071515A1 (fr) * 1981-07-30 1983-02-09 Laboratoires de Therapeutique Moderne L.T.M. Procédé d'obtention de polyosides capsulaires, polyosides capsulaires ainsi obtenus et leur application à la préparation de vaccins
WO1994009115A1 (fr) * 1992-10-15 1994-04-28 Merck & Co., Inc. PROCEDE DE CROISSANCE CLONALE DE $i(STREPTOCOCCUS PNEUMONIAE)
WO2004083251A2 (fr) 2003-03-17 2004-09-30 Wyeth Holdings Corporation Holotoxine du cholera mutante en tant qu'adjuvant et proteine de support d'antigene
WO2010080484A1 (fr) * 2008-12-18 2010-07-15 Wyeth Llc Procédé de contrôle du poids moléculaire d'un polysaccharide provenant de streptococcus pneumoniae en utilisant du carbone

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
C. LIBERMAN, J IND MICROBIOL BIOTECHNOL, vol. 35, 2008, pages 1441 - 1445
GONÇALVES V M ET AL: "Optimization of medium and cultivation conditions for capsular polysaccharide production by Streptococcus pneumoniae serotype 23F", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER VERLAG, BERLIN, DE, vol. 59, no. 6, 1 September 2002 (2002-09-01), pages 713 - 717, XP002569808, ISSN: 0175-7598, [retrieved on 20040219], DOI: 10.1007/S00253-002-1075-8 *
LIBERMAN C ET AL: "Pneumococcal whole-cell vaccine: optimization of cell growth of unencapsulated Streptococcus pneumoniae in bioreactor using animal-free medium", JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY ; OFFICIAL JOURNAL OF THE SOCIETY FOR INDUSTRIAL MICROBIOLOGY, SPRINGER, BERLIN, DE, vol. 35, no. 11, 20 August 2008 (2008-08-20), pages 1441 - 1445, XP019637539, ISSN: 1476-5535, DOI: 10.1007/S10295-008-0445-3 *
V.M. GONÇALVES, APPL MICROBIOL BIOTECHNOL, vol. 59, 2002, pages 713 - 717

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014038879A1 (fr) * 2012-09-07 2014-03-13 에스케이케미칼주식회사 Procédé de production d'un polysaccharide de capsule ayant un sérotype pneumococcique
US9636392B2 (en) 2012-09-07 2017-05-02 Sk Chemical Co., Ltd. Production method for capsular polysaccharide having pneumococcal serotype
WO2016207744A1 (fr) * 2015-06-26 2016-12-29 Biobridge Healthcare Solutions Pvt. Ltd. Procédé amélioré pour la production et la purification de polysaccharide pneumococcique capsulaire de streptococcus pneumoniae et procédé de production de composition de vaccin conjugué utilisant ledit polysaccharide
AU2016357567B2 (en) * 2015-11-17 2020-05-07 Pfizer Inc. Media and fermentation methods for producing polysaccharides in bacterial cell culture
JP2022017529A (ja) * 2015-11-17 2022-01-25 ファイザー・インク 細菌細胞培養物中で多糖を生成するための培地および発酵方法
KR20180073699A (ko) * 2015-11-17 2018-07-02 화이자 인코포레이티드 박테리아 세포 배양에서 폴리사카라이드를 생성하기 위한 배지 및 발효 방법
CN108473945A (zh) * 2015-11-17 2018-08-31 辉瑞公司 用于在细菌细胞培养物中生产多糖的培养基和发酵方法
JP2018533366A (ja) * 2015-11-17 2018-11-15 ファイザー・インク 細菌細胞培養物中で多糖を生成するための培地および発酵方法
US20190292513A1 (en) * 2015-11-17 2019-09-26 Pfizer Inc. Media and fermentation methods for producing polysaccharides in bacterial cell culture
KR102069988B1 (ko) * 2015-11-17 2020-01-23 화이자 인코포레이티드 박테리아 세포 배양에서 폴리사카라이드를 생성하기 위한 배지 및 발효 방법
WO2017085602A1 (fr) * 2015-11-17 2017-05-26 Pfizer Inc. Milieux et procédés de fermentation pour la production de polysaccharides dans une culture de cellules bactériennes
US10947494B2 (en) 2015-11-17 2021-03-16 Pfizer Inc. Media and fermentation methods for producing polysaccharides in bacterial cell culture
JP7311966B2 (ja) 2015-11-17 2023-07-20 ファイザー・インク 細菌細胞培養物中で多糖を生成するための培地および発酵方法
RU2761633C2 (ru) * 2015-11-17 2021-12-13 Пфайзер Инк. Среды и способы ферментации для производства полисахаридов в бактериальной клеточной культуре
IL265623B (en) * 2016-09-30 2022-12-01 Biological E Ltd Multivalent vaccines for lung spot that include polysaccharide-protein conjugates
US11547752B2 (en) 2016-09-30 2023-01-10 Biological E Limited Multivalent pneumococcal vaccine compositions comprising polysaccharide-protein conjugates
IL297575B1 (en) * 2016-09-30 2023-08-01 Biological E Ltd Multivalent vaccines for lung spot that include polysaccharide-protein conjugates
CN107723337A (zh) * 2017-11-30 2018-02-23 成都欧林生物科技股份有限公司 筛选肺炎链球菌用固体培养基的制备方法
CN115335504A (zh) * 2019-11-18 2022-11-11 Vaxcyte公司 链球菌的无动物产物培养
WO2021101904A1 (fr) * 2019-11-18 2021-05-27 Vaxcyte, Inc. Culture sans produit animal de bactéries streptococcus
CN117821342A (zh) * 2024-02-29 2024-04-05 北京民海生物科技有限公司 一种14型肺炎链球菌的发酵方法
CN117821342B (zh) * 2024-02-29 2024-06-04 北京民海生物科技有限公司 一种14型肺炎链球菌的发酵方法

Also Published As

Publication number Publication date
AR082374A1 (es) 2012-12-05

Similar Documents

Publication Publication Date Title
JP5465676B2 (ja) 連鎖球菌属を培養するための発酵プロセスおよび連鎖球菌属由来の莢膜多糖（ｃｐ）を得るための精製プロセス
WO2011151841A1 (fr) Procédé de fermentation pour streptococcus pneumoniae
EP2922569B1 (fr) Production de hauts rendements de polysaccharides bactériens
KR101341121B1 (ko) 디프테리아 독소를 생성하기 위한 발효 방법
CN108473945A (zh) 用于在细菌细胞培养物中生产多糖的培养基和发酵方法
KR101161033B1 (ko) 컨쥬게이트 백신에 사용하기 위한 피막 다당류의 제조 방법
CN104487086B (zh) 无动物源的不含酒精的疫苗组合物及其制备方法
KR102666639B1 (ko) 대장균 조성물 및 이의 사용 방법
AU2013313794A1 (en) Production method for capsular polysaccharide having pneumococcal serotype
WO2016207744A1 (fr) Procédé amélioré pour la production et la purification de polysaccharide pneumococcique capsulaire de streptococcus pneumoniae et procédé de production de composition de vaccin conjugué utilisant ledit polysaccharide
KR102028693B1 (ko) 스트렙토코커스 뉴모니아 협막 다당체의 생산방법
CN103833865B (zh) 一种肺炎链球菌荚膜多糖的制备方法
Liberman et al. Use of soy peptones for Streptococcus pneumoniae cultivation for vaccine production
RU2788526C2 (ru) Композиции escherichia coli и способы их применения
KR20240001048A (ko) 이. 콜라이 FimH 돌연변이체 및 그의 용도
EP2942396A1 (fr) Polysaccharides produits par mutants CPSC
MXPA06002813A (en) Process for producing polysaccharide for conjugate vaccine

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11738497

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11738497

Country of ref document: EP

Kind code of ref document: A1