Preparation method of acellular pertussis vaccine

A pertussis and toxin technology, which is applied in the field of acellular pertussis vaccine preparation, can solve the problems of difficult to exceed 70% yield, conflict between purity and yield, existence of safety, etc., and achieves easy large-scale scale-up production and good stability. , the effect of high load

Active Publication Date: 2020-03-17
CHANGCHUN BCHT BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But this product has some disadvantages: the side reaction is relatively large, and there are still certain problems in safety.
[0014] Furthermore, although these methods can relatively easily carry out quality control on the ratio of each antigenic component, etc., there is a conflict between purity and yield, especially when the PT purity reaches more than 90%, the yield is difficult to exceed 70%.

Method used

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  • Preparation method of acellular pertussis vaccine
  • Preparation method of acellular pertussis vaccine
  • Preparation method of acellular pertussis vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1. Fermentation of Bacillus pertussis

[0063] experiment material:

[0064] Species: Bordetella pertussis CS phase I strain

[0065] Medium: Improved SS liquid medium: SS liquid medium with 0.25% acid hydrolyzed casamino acids and 0.1% β-cyclodextrin

[0066] Rehydration: sodium glutamate 4g / L, acid hydrolyzed casamino acids 2g / L, pH7.6

[0067] Main equipment: 40L fermenter (BIO-FLOW5000, NBS), 300L fermenter (BIOF-6300B 300L, Shanghai Gaoji Biological Engineering Co., Ltd.)

[0068] experiment method:

[0069] Put the freeze-dried bacterial species of pertussis CS strain on sheep blood abalone ginger agar medium containing 25% defibrillation, culture at 37°C for 72 hours, transfer to activated carbon agar medium, and cultivate at 37°C for 48 hours; pass 3 Generation (activated carbon agar medium), after continuing to culture at 37°C for 48 hours, scrape seeds in 150ml phosphate buffer (ie PBS buffer, 137mM NaCl, 2.7mMKCl, 10mMNa 2 HPO 4 , 2mM KH 2 PO ...

Embodiment 2

[0072] Embodiment 2. The ultrafiltration treatment of fermented liquid

[0073] Test materials and samples:

[0074] Hollow fiber filter column: UFP-10 / 30-C-4MA-0.065m2, GE

[0075] Tangential flow treatment system: Quixstand hollow fiber membrane separation system, GE

[0076] Sample: Bacterial fluid harvested from pertussis fermentation, supernatant after centrifugation or other methods to separate the bacterial cells, and filtered through a 0.22 μm filter membrane to remove impurities. The sample volume is 3000ml.

[0077] experiment method:

[0078] Add phosphate buffer (pH8.0) and urea to the supernatant after 3000ml bacterial cell separation to make the final concentration respectively 50mmol / l and 1mol / l, as the protective agent in the ultrafiltration process. The supernatant liquid added with the protective agent was concentrated by ultrafiltration at a flow rate of 4000 / sec using a hollow fiber column with a molecular weight cut-off of 10KD / 30KD, and the pressure a...

Embodiment 3

[0081] Example 3. Purification of pertussis effective antigen (1)

[0082] Test materials and samples:

[0083] Chromatographic column: XK26×20, GE

[0084] UV detector: UVD-680-1, Shanghai Jinda Biochemical Instrument Co., Ltd.

[0085] Peristaltic pump: BT-300J, Baoding Lange Constant Flow Pump Co., Ltd.

[0086] Ion exchange packing: CM-Sepharose, GE; Capto-adhere, GE

[0087] Gel filtration filler: Sephacryl S-200 High Resolution

[0088] Sample: 200ml of harvest liquid after ultrafiltration and concentration treatment

[0089] Purification of PT and FHA:

[0090] Put 60ml of CM-Sepharose medium into a 2.6×20CM chromatographic column, and equilibrate 3-5 column volumes with pH6.02M urea and 50mM phosphate buffer. Take 200ml of the supernatant obtained by ultrafiltration, pass the liquid through the chromatographic column at a flow rate of 300ml / h, monitor the protein A280 absorption value with ultraviolet light, and collect the flow-through at the same time to separa...

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Abstract

The invention relates to a method for preparing an acellular pertussis vaccine, comparing: ultrafiltrating and condensing bordetella pertussis culturing supernatant, to obtain a concentrated solution; carrying out cation exchange column chromatography on the concentrated solution, to separate out pertussis toxin (PT) and filamentous hemagglutinin (FHA); optionally, carrying out multi-combination anion exchange chromatography on a flowing-through liquid obtained in a step (2), to separate out pertactin (Prn); PT is individually used to prepare the acellular pertussis vaccine, or PT is mixed with FHA and / or Prn to prepare the acellular pertussis vaccine.

Description

technical field [0001] The invention relates to a preparation method of pertussis vaccine and pertussis vaccine. More specifically, the present invention relates to the separation and purification of effective antigenic components of pertussis, the preparation method of acellular pertussis vaccine and the acellular pertussis vaccine. Background technique [0002] Pertussis vaccine is the most effective and economical way to prevent the disease, which includes whole cell pertussis vaccine (WPV) and acellular pertussis vaccine (acellular pertussis vaccine, APV). [0003] The whole-cell pertussis vaccine was successfully developed for the first time in 1914. It was sterilized by appropriate methods after mass culture and enrichment of bacteria to inactivate them into non-infectious but good immunogenic whole pertussis bacteria, and then added adjuvant preparations. In controlling and reducing the incidence of whooping cough in children, the whole-cell pertussis vaccine has pl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/235C07K1/36C07K1/34C07K1/18A61K39/10A61P31/04
Inventor 王雪云王长永谷义平徐艳艳姜涛曾红柏建力崔善海夏富文王洪飞
Owner CHANGCHUN BCHT BIOTECH
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