WO2019138432A1 - Composition de milieu pour la production de polysaccharides bactériens - Google Patents

Composition de milieu pour la production de polysaccharides bactériens Download PDF

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Publication number
WO2019138432A1
WO2019138432A1 PCT/IN2019/050037 IN2019050037W WO2019138432A1 WO 2019138432 A1 WO2019138432 A1 WO 2019138432A1 IN 2019050037 W IN2019050037 W IN 2019050037W WO 2019138432 A1 WO2019138432 A1 WO 2019138432A1
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WO
WIPO (PCT)
Prior art keywords
production
neisseria meningitidis
polysaccharides
fermentation
media
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PCT/IN2019/050037
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English (en)
Inventor
Sandeep Sharma
Nitin Kumar
Sarmad HANIF
Manoj Kumar CHHIKARA
Davinder Gill
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Msd Wellcome Trust Hilleman Laboratories Pvt. Ltd.
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Application filed by Msd Wellcome Trust Hilleman Laboratories Pvt. Ltd. filed Critical Msd Wellcome Trust Hilleman Laboratories Pvt. Ltd.
Publication of WO2019138432A1 publication Critical patent/WO2019138432A1/fr

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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Definitions

  • the present invention relates to production of bacterial polysaccharides.
  • the present invention particularly relates to culture media composition, feed composition and fermentation conditions for production of Neisseria meningitidis polysaccharides.
  • the N. meningitidis polysaccharides of the present invention are capable of being used in the production of economical polysaccharide protein conjugate vaccine(s) against meningococcal infections.
  • Neisseria meningitidis is a Gram- negative bacterium that can cause meningitis and other forms of meningococcal disease such as meningococcemia.
  • N. meningitidis On the basis of the type of capsular polysaccharide present on N. meningitidis (Men), thirteen serogroups have been identified and among the 13 identified capsular types of N. meningitidis, six (A, B, C, W135, X, and Y) account for most meningococcal disease cases worldwide. MenA has been the most prevalent serogroup in Africa and Asia but is rare/ practically absent in North America. In Europe and United States, serogroup B (MenB) is the predominant cause of disease and mortality, followed by serogroup MenC and MenW. In recent past, MenX outbreaks have started showing up in sub-Saharan Africa. The multiple serogroups have hindered development of a universal vaccine for meningococcal disease.
  • the immune response may be characterised as T-cell dependent (TD) immune response and T-cell independent (TI) immune response.
  • TD T-cell dependent
  • TI T-cell independent
  • Proteins and peptides are known to elicit TD antigens by stimulating the helper T lymphocytes and generating memory cells.
  • polysaccharides belong to the TI antigens which do not induce T-cell activation and do not form any memory B cells, which is a major drawback while dealing with infants as they have an immature immune system.
  • the polysaccharides, especially antigenic polysaccharides, used in preparation of vaccines may be monovalent, bivalent and poly (multi) valent vaccines containing one, two or more polysaccharides, respectively.
  • the multivalent polysaccharide based vaccines have been used for many years and have proved valuable in preventing diseases such as Pneumococcal, Meningococcal or Haemophilus influenzae diseases.
  • the production of purified N. meningitidis capsular polysaccharides is the foremost requirement for an effective conjugation with the carrier protein and its development as a conjugate vaccine.
  • the cost for the cultivation of N. meningitidis for production of capsular polysaccharides is generally high and involves long working hours since it involves a series of production and quality control steps. An optimized medium/process can obviate these issues.
  • patent application no. US 12/041,745 discloses a method of producing a meningococcal meningitis vaccine, the method, includes culturing N. meningitidis to produce capsular polysaccharides of serogroups A, C, Y and W-135 in N. meningitidis fastidious medium (NMFM), isolating the capsular polysaccharides from the culture, purifying the capsular polysaccharides of any residual cellular biomass; and depolym prizing the capsular polysaccharide mechanically.
  • NMFM meningitidis fastidious medium
  • the cited art utilizes long hours for the production of purified capsular polysaccharide.
  • Another US patent publication no. US 20150299750 A1 discloses an improved culture, fermentation and purification conditions for preparing Neisseria meningitidis polysaccharides.
  • Another US patent publication no.: 20080318285 A1 discloses Neisseria meningitidis fastidious medium designed to maximize the yield of capsular polysaccharides and generate minimal cellular bio mass and endotoxin in a short duration of fermentation.
  • the main object of the present invention is to provide a process of production of bacterial polysaccharide.
  • Another object of the present invention is to provide a process of production of capsular polysaccharides of various serogroups of Neisseria meningitidis.
  • Yet another object of the present invention is to provide optimized culture media and feed media composition for better growth. Yet another object of the present invention is to provide improved culture media composition for growth of fastidious Neisseria meningitidis serogroups A, C, W, and Y. Yet another object of the present invention is to provide improved feed media composition for growth of fastidious Neisseria meningitidis serogroups A, C, W, and Y. Yet another object of the present invention is to provide process of fermentation in reduced time with better polysaccharide yield with low impurities in a very short time by simple, efficient, improved and commercially scalable methods. Yet another object of the present invention is to produce high quality product with better yield that meet the relevant quality specifications.
  • the present invention describes a rapid, industrially scalable, cost effective process for growth of bacteria preferably Neisseria meningitidis for production of bacterial polysaccharide.
  • the present invention describes culture media for N. meningitidis including but not limited to di-sodium hydrogen phosphate in the range of 3.00 ⁇ 1.0 g/L, potassium phosphate monobasic in the range of 0.8125 ⁇ 0.01 g/L, L-cystine in the range of 0.05 ⁇ 0.01 g/L, Magnesium sulphate in the range of 0.3 ⁇ 0.1 g/L, beta alanine in the range of 0.025 ⁇ 0.01 g/L, L- glutamic acid in the range of 1.5 ⁇ 0.25 g/L, casamino acid in the range of 12.00 ⁇ 2 g/L, yeast extract in the range of 10.00 ⁇ 2 g/L and dextrose in the range of 10.00 ⁇ 2.0 g/L,
  • the above-mentioned culture media composition provides optimal growth for N. meningitidis serogroups.
  • the present invention also describes feed media for N. meningitidis including but not limited to L-glutamic acid in the range of 8.00 ⁇ 2.0g/L, dextrose in the range of 25 ⁇ 2.0 g/L, soya peptone in the range of 20 ⁇ 2.0 g/L and other components like ammonium chloride as per the requirement.
  • feed media for N. meningitidis including but not limited to L-glutamic acid in the range of 8.00 ⁇ 2.0g/L, dextrose in the range of 25 ⁇ 2.0 g/L, soya peptone in the range of 20 ⁇ 2.0 g/L and other components like ammonium chloride as per the requirement.
  • the above-mentioned feed media composition provides optimal growth for N. meningitidis serogroups when added in the fermentation broth during fermenter culture with the aforementioned culture media.
  • the present invention describes the fermentation process at predetermined temperature, pH, airflow, dissolved oxygen and rate of agitation, such that the fermentation is completed within 11 ⁇ 3 hours.
  • Figure-1 depicts Shake Flask studies for media optimization using MenW (4 media compositions)
  • Figure-3 depicts growth curves of MenA
  • Figure-4 depicts growth curves of MenC
  • Figure-5 depicts growth curves of MenY
  • Figure-6 depicts growth curves of MenW
  • Figure-7 depicts NMR profile of MenA
  • the present invention discloses a process of production of bacterial polysaccharides.
  • the present invention particularly relates to optimized culture media and feed media for growth of fastidious Neisseria meningitidis in lesser time.
  • the invention also relates to fermentation conditions for production of Neisseria meningitidis polysaccharides.
  • the N. meningitidis polysaccharides of the present invention are capable of being used in the production of economical polysaccharide protein conjugate vaccine(s) against meningococcal infections.
  • the present invention describes culture media for N. meningitidis comprising sodium phosphate dibasic in a concentration of 3.00 g/L, potassium phosphate monobasic in the concentration of 0.8125 g/L, L-cystine in the concentration of 0.05 g/L, magnesium sulphate in the concentration of 0.3 g/L, beta alanine in the concentration of 0.025 g/L, L-glutamic acid in the concentration of 1.5. g/L, casamino acid in the concentration of 12.0 g/L, yeast extract in the concentration of 10 g/L, dextrose in the concentration of 10 g/L and ammonium chloride in the concentration of 2.00 g/L.
  • the above-mentioned culture media composition provides optimal growth for N. meningitidis serogroups MenA, MenC, MenY and MenW. Ammonium chloride is added only to Serogroups W for optimal polysaccharide production.
  • the present invention also describes feed media for N. meningitidis including but not limited to L-glutamic acid in the range of 8.00 ⁇ 2.0g/L, dextrose in the range of 25.00 ⁇ 2.0 g/L and soya peptone in the range of 20.00 ⁇ 2.0 g/L.
  • the above-mentioned feed media composition provides optimal growth for N. meningitidis serogroups.
  • the above-mentioned feed media composition provides optimal growth for N. meningitidis serogroups Men A, MenC, MenY and MenW.
  • the optimized feed composition is listed in Table 3 of the specification.
  • Example 5 After growth of bacteria in flask with optimized culture media, the bacteria are subjected to fermentation as disclosed in Example 5 and Example 6 of the specification.
  • the fermentation conditions are so optimized that the resultant fermentation harvest (broth) have high polysaccharide yield and low level of impurities and the fermentation process is completed within 11 ⁇ 3 hours, preferably 10 to 12 hours.
  • the fermentation is carried out in a temperature range of 36 ⁇ 1° C with rpm in the range of 100 to 700 rpm, the air flow of the fermenter is maintained at 0.2 to 1.2. 1/ m and the partial pressure of Oxygen (PO2) is maintained from 100% to 15% during the course of fermentation along with a pH of 7.3 ⁇ 0.1.
  • the present invention provides a rapid, industrially scalable, cost effective process for the production of Neisseria meningitidis serogroups MenA, MenC, MenY and MenW with optimized culture media and feed media which provides maximum growth to the Neisseria meningitidis.
  • Example-1 Shake Flask experiments for media optimization using MenW
  • Inhibition ELISA method is used for estimation of the polysaccharide content in the bacterial culture broth.
  • the sample containing meningococcal capsular polysaccharide is incubated with the serogroup specific polyclonal antibody (primary antibody) so that complexes will be formed between the antibody and antigens in the sample.
  • primary antibody the serogroup specific polyclonal antibody
  • These complexes are then added to a container in which competitor homologous antigens are immobilized.
  • Antibody which is not complexed with immunogens from the polysaccharide test sample bind to these immobilized competitor antigens.
  • the antibody which is bound to the immobilized competitor antigens (after usual washing steps, etc.) can then be detected by adding an enzyme labelled secondary antibody which binds to the primary antibody.
  • the label is used to identify the reaction of immobilized primary antibody to secondary antibody utilizing a chromogenic substrate.
  • the reduction in the absorbance in test well as compared to the control well (without any test sample) confirms the presence of the specific antigen in the test sample and the percentage inhibition of the antibody is directly proportional to the polysaccharide concentration in the test sample.
  • the ELISA is performed, wherein the Plate A is coated with IOOmI of coating solution having equal volume of in-house PS and mHSA and incubated for overnight at 2-8°C. A no-antigen-control is included as control.
  • the coated plate is blocked at room temperature with 200m1 of blocking buffer.
  • Quality control polysaccharide (Standard) of defined concentration are serially diluted three-fold as are the bacterial culture supernatant (test samples) and incubated in Plate B with serogroup specific polyclonal primary antibody for 1 hour at 37°C.
  • the antigen- antibody mixture from Plate B is transferred to blocked Plate A and further incubated for two hours (1.5 hours at 37°C and half an hour at room temperature).
  • the plate is further incubated with secondary antibody for 1 hour and reaction is developed using IOOmI of TMB substrate and incubated for 10 min.
  • the reaction is stopped with 50m1 of 2M H2SO4 per well before OD at 450 nm is observed with reference to 630nm.
  • the inhibition percentage is calculated from inhibition of OD in standard or test sample dilutions in relation to OD of no-antigen control wells.
  • the standard curve is generated from inhibition percentages for quality control dilutions which is used to extrapolate the concentration of polysaccharide in the test samples using Combistat software ( Figure- 1).
  • Both media compositions 2 and 3 are merged and are finally selected and taken forward for scale-up/ fermentation experiments (2.5L scale) each for Men A, C, Y and W serogroups. The growth of all the serogroups is depicted in Figures 3-6.
  • the final fermenter media composition is described in Table-2 below.
  • Example-4 Feed composition
  • the Feed composition is finalized during the fermentation experiments and is described in Table-3 for the production of Men A, C, Y and W. Furthermore to achieve higher growth for MenW, ammonium chloride is added at the 10th hour of fermentation in the range of 2 ⁇ 0.2 g/L in the feed media
  • the above-mentioned feed composition as listed in Table-3 is unique and supports better growth of all serogroups (Men A, MenC , MenY, and MenW).
  • the nutrient fermentation media and feed components utilized in the present invention are cost effective, simple and lead to low cellular biomass production with low levels of endotoxins and thus result in polysaccharide which has minimal level of impurities in the harvested fermentation broth.
  • One WCB vial is withdrawn from deep freezer and transfered into mini cooler.
  • the vial is thawed inside the biosafety cabinet and one modified GC agar plate is aseptically streaked with the help of inoculation loop. Afterwards, the plate is incubated at 36 ⁇ 1°C with 5 ⁇ 0.5% CO2 for 24 to 36 hours. Colony morphology is checked on modified GC agar plate and Gram staining is done to check the purity. Gram negative, non-spore forming, non- motile, encapsulated, diplococci, appeared under the microscope.
  • one flask is inoculated with 4-5 colonies and incubated at 36 ⁇ 1°C with 5 ⁇ 0.5% CO2 at 150 ⁇ 5 rpm for 4 to 6 hours. After 4 to 6 hours OD at 550 nm and purity by Gram staining is checked. When the growth reaches an ODsso nm of 1.00 ⁇ 0.1, the flask culture is aseptically inoculated into the fermenter.
  • p02 tends to decrease. Maintain the p02 at 30% for initial 6 hours of fermentation, followed by 20% p02 from 7th hour to 8th hour of fermentation and 15% p02 from 8th hour of fermentation till the end of fermentation run by increasing rpm and airflow. If the p02 is not controlled by rpm, compressed air is replaced with pure oxygen. OD (at 550nm) ⁇ and purity of culture is checked after every 2 hours. As soon as the OD at 550nm of culture reaches 1 ⁇ 0.1 the addition of feed is initiated at a flow rate of 1 ml/ min. The fermentation is carried out in optimized conditions as enumerated in Table 4 below:
  • Example 8 Purified PS yields for MenA, C, Y and W
  • the present invention provides improved culture and feed media, for better production of N. meningitidis polysaccharides by fermentation in reduced time with high yields.

Abstract

La présente invention concerne une composition de milieu de culture, une composition d'alimentation et des conditions de fermentation pour la production de polysaccharides de Neisseria meningitidis. Les polysaccharides de N. meningitidis de la présente invention peuvent être utilisés dans la production d'un ou de plusieurs vaccins conjugués protéine-polysaccharide économiques contre des infections méningococciques.
PCT/IN2019/050037 2018-01-15 2019-01-15 Composition de milieu pour la production de polysaccharides bactériens WO2019138432A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115478034A (zh) * 2022-10-13 2022-12-16 艾美卫信生物药业（浙江）有限公司 一种脑膜炎球菌发酵培养方法、发酵液、精制多糖、应用

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2005103230A2 (fr) * 2004-04-22 2005-11-03 Chiron Srl Peptone de soja servant de source d'azote pour preparer des conjugues meningococciques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005103230A2 (fr) * 2004-04-22 2005-11-03 Chiron Srl Peptone de soja servant de source d'azote pour preparer des conjugues meningococciques

Non-Patent Citations (2)

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KAMPEN, W.H. ET AL.: "Nutritional requirements in fermentation processes", IN FERMENTATION AND BIOCHEMICAL ENGINEERING HANDBOOK, 2014, Norwich, NY, USA, pages 37 - 57 *
SHARMA S ET AL.: "Rapid Process for purification of capsular polysaccharide from Neisseria meningitidis serogroups A and C", BIOLOGICALS, vol. 43, 27 June 2015 (2015-06-27), pages 383 - 389, XP029269960 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115478034A (zh) * 2022-10-13 2022-12-16 艾美卫信生物药业（浙江）有限公司 一种脑膜炎球菌发酵培养方法、发酵液、精制多糖、应用
CN115478034B (zh) * 2022-10-13 2023-04-18 艾美卫信生物药业（浙江）有限公司 一种脑膜炎球菌发酵培养方法、发酵液、精制多糖、应用

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