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Methods of cultivating bordetella species

A technology of Bordetella and Tertella, applied in biochemical equipment and methods, bacteria, antibacterial drugs, etc., can solve problems such as antigen loss

Pending Publication Date: 2021-03-16
圣诺菲·帕斯图尔公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, a substantial portion of the PRN usually remains associated with the cells, and as such, it is absent from the supernatant, leading to a potential loss of antigen

Method used

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  • Methods of cultivating bordetella species
  • Methods of cultivating bordetella species
  • Methods of cultivating bordetella species

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1. Strong acids increase PRN production in supernatants from Bordetella cultures.

[0103] Working seeds (2.5 mL) were inoculated in 2L shake flasks each containing 500 mL of broth (modified Stainer-Scholte medium, Table 3) with 5 mL of 100x growth factor solution (Table 4). The culture was grown in an incubator shaker for 44 hours and used to inoculate a 2L fermentor / bioreactor (2L BIOSTAT B fermenter (B. Braun Biotech International, Berlin, Germany)) containing 1150 mL of a solution supplemented with growth factors ( Table 4) broth (modified Stainer-Scholte medium, Table 3).

[0104] Feed supplements of monosodium glutamate and growth factors, glutathione, ferrous sulfate, ascorbic acid, niacin, and cysteine ​​are continuously added to the bioreactor during the incubation period to increase antigen production . The addition of supplemental solution was triggered by a sharp increase in dissolved oxygen (DO) levels and a sharp decrease in agitation, which occu...

Embodiment 2

[0115] Example 2. Acid mixtures increase PRN and FIM2 / 3 production in supernatants from Bordetella cultures while mitigating PT and FHA loss.

[0116] Cultures were prepared as described above for Example 1, except that different ratios of mixtures of nitric acid and phosphoric acid were used for bioreactor pH control. PRN, PT, FHA, and FIM2 / 3 antigens were isolated to assess the effect of the acid mixture on antigen production. At the end of the incubation period, the harvest was centrifuged to obtain a cell fraction and a supernatant fraction. Supernatant fractions were assayed for PRN, PT, FHA and FIM2 / 3 antigens by ELISA. Cell pellets were resuspended in water for injection (WFI) and treated with 8M urea to lyse cells and release FIM2 / 3 for analysis by ELISA.

[0117] Figures 2A-2D The production of each antigen in the bioreactor harvest as measured by ELISA is shown for multiple bioreactor runs. Figure 2A It was confirmed that PRN production in the bioreactor harves...

Embodiment 3

[0120] Example 3. Acid mixtures and chemically defined media further enhanced PRN and FIM2 / 3 production in supernatants from Bordetella cultures while mitigating PT and FHA loss.

[0121] Cultures were also prepared as described above for Example 1, except chemically defined medium was used instead of complex medium. As described in Example 2, a mixture of nitric acid and phosphoric acid at different ratios was used for bioreactor pH control. PRN, PT, FHA and FIM2 / 3 antigens were isolated as described above for Example 2.

[0122] Figure 3 shows the production of each antigen in the bioreactor harvest, as measured by ELISA, for multiple bioreactor runs. Figure 3A -D also confirmed that when 100% 3.5M nitric acid was used instead of 100% 2.5M phosphoric acid for pH control, the yield of PRN in the bioreactor harvest supernatant fraction increased (5-7mg / L to 14-22mg / L L). Additionally, Figure 3 also confirms that when a solution containing 100% 3.5M nitric acid was used inste...

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Abstract

The present disclosure is directed to a method for cultivating a Bordetella species, comprising: cultivating a Bordetella species under aerobic conditions in a liquid culture medium; and maintaining apH of the liquid culture medium by using a strong acid, such as nitric acid, or using a first and second acid, wherein the first acid is an inorganic acid that dissociates essentially completely in water, such as nitric acid, hydrochloric acid or sulfuric acid, and wherein the second acid is an inorganic acid having an acid dissociation constant (pKa) of greater than 1, such as phosphoric acid. Methods for increasing the yield of Bordetella fimbrial agglutinogen 2 and fimbrial agglutinogen 3 (FIM2 / 3) in a supernatant fraction from a Bordetella culture are also provided.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of and relies on the filing date of U.S. Provisional Patent Application No. 62 / 645,473, filed March 20, 2018, the entire disclosure of which is incorporated herein by reference. technical field [0003] The present application generally relates to methods for growing Bordetella species such as Bordetella pertussis. Background technique [0004] Whooping cough is a bacterial infection of the lungs characterized by severe coughing attacks. Bordetella pertussis is the main causative agent of this condition. Vaccines (wP) comprising inactivated whole cells of B. pertussis, chemically detoxified and formulated with diphtheria and tetanus antigens have been available for decades. Since the 1990s, the wP vaccine has been replaced by the acellular pertussis vaccine in many countries. Compared with the wP vaccine, the acellular pertussis (aP) vaccine induces relatively few side effects ass...

Claims

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Application Information

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IPC IPC(8): C12N1/20A61K39/10A61P31/04A61P37/04C12N1/38C12P21/08
CPCC12N1/38A61P37/04C12N1/20A61P31/04C07K14/235A61K39/099C12N2500/02C12N2500/34C12N2500/60C12N2500/72
Inventor P·法雷尔B·Z·孙F·巴比拉托J·D·J·梅内德斯·迪亚兹A·查佩塔
Owner 圣诺菲·帕斯图尔公司
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