Haemophilus influenzae Type b Conjugate Vaccine
Haemophilus influenzae Type b Conjugate Vaccine is a preparation made by covalently binding the purified capsular polysaccharide (CPS) antigen from Haemophilus influenzae Type b to tetanus toxoid through the linkage of adipic dihydrazide (ADH). It is used to prevent childhood infectious diseases caused by Haemophilus influenzae Type b, such as cerebrospinal meningitis, and pneumonia, etc.
1 Basic requirement
The facilities, source and subsidiary materials, water, apparatus, and animals used for production and quality control shall comply with the requirements set forth in the General Notices. 2 Manufacturing 2.1 Bacterial seed
The bacterial seed used for production complies with the Requirements for Bacterial and Viral strains/seeds Used for Production and Quality Control of Biologics. 2.1.1 Name and origin of bacterial strains
The bacterial strain used for production shall be Haemophilus influenzae type b strain CMCC58547 or CMCC58534. 2.1.2 Establishment of seed lot system
It complies with the Requirements for Bacterial and Viral strains/seeds Used for Production and Quality Control of Biologics. 2.1.3 Passage of seed lots
The subculture of the seed from master seed lot to working seed lot shall not exceed five passages; the subculture of the seed from working seed lot to the final fermentation shall not exceed five passages. 2.1.4 Control tests on the seed lots
Chocolate agar medium containing sheep blood or Hib comprehensive medium can be used for the control tests on the seed lots. 2.1.4.1 Cultural characteristics
The Hib strain shall not grow on a common agar medium, but grow on a chocolate agar medium containing sheep blood or a Hib comprehensive medium. Its growth requires Factor X ( chlorhematin ) and Factor V ( β coenzyme A ); the satellite phenomenon shall be positive; the colonies shall be greyish-white, translucent, smooth, convex and humid with regular edge. 2.1.4.2 Microscopic examination of stained smears
The stained smears made from the culture shall be Gram-negative capsulated coccobacillus, sometimes in short chain, or single Gram-negative coccus. 2.1.4.3 Biochemical reactions
The culture shall ferment glucose, xylose and galactose, with the production of acid but no gas. They shall not ferment sucrose, lactose, or fructose. The lysine decarboxylase reaction shall be negative.
2.1.4.4 Serological test
After incubation at 35-37℃, take the lawn to perform a slide agglutination test with Hib antiserum, It shall give a strong agglutination.
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2.1.5 Storage of seed lots
Seed lots shall be lyophilized and stored at or below 8℃. 2.2 Bulk 2.2.1 Seeds for production
After cultural characteristics and microscopic examination of stained smears are examined as qualified, the bacterial seeds derived from working seed lot shall be inoculated onto liquid or solid Hib comprehensive medium to prepare a quantity of seeds for production. 2.2.2 Culture medium for production
The Hib comprehensive medium shall be used for production. 2.2.3 Cultivation
Inoculate the bacterial seeds into liquid medium in a fermentor, Take samples during cultivation and test for bacterial purity by microscopic examination of Gram-stained smear,. If any contaminating microorganisms are found, the cultures shall be discarded. 2.2.4 Harvest and killing of the organisms
The cultures shall be harvested during the late logarithmic of early stationary phase. Take samples to test for bacterial concentration and purity. The qualified bacterial culture shall be killed by addition of formaldehyde solution or sodium deoxycholate in adequate concentration. It is proper to ensure complete killing of bacteria without damage to Hib capsular polysaccharide. 2.2.5 Extraction and purification of polysaccharide 2.2.5.1. Removal lf nucleic acid
After killing of the bacteria, centrifuge the culture, remove the bacteria and collect the supernatant. Add hexadecyl trimethylammonium bromide to the supernatant to form a precipitate and collect combined polysaccharide.
2.2.5.2. Precipitation of polysaccharide
Collect crude polysaccharide by ethanol precipitation. Alternatively, concentrate by ultrafiltration and precipitate by ethanol, take the supernatant, add ethanol in an appropriate concentration to precipitate the polysaccharide, and wash the precipitate with absolute ethanol and acetone successively, to prepare crude polysaccharide after drying. Store at or below -20℃. 2.2.5.3. Purification of polysaccharide
Dissolve the crude polysaccharide in 1/10 saturated sodium saturated sodium acetate solution, then extract several times with cold phenol in a proper ratio. Collect the supernatant, and remove phenol by dialysis or ultrafiltration. Then add ethanol to a final concentration of 60%-80%, collect the precipitate by centrifugation. Alternatively, concentrate by ultrafiltration and precipitate by ethanol, take the supernatant, then add ethanol to a final concentration of 60%-80%. Wash it with absolute ethanol and acetone respectively. The solid after drying under vacuum is purified polysaccharide.
2.2.5.4. Control tests on the purified polysaccharide See Section 3.1.
2.2.5.5. Storage and storage period
The purified polysaccharide shall be stored at or below -20℃. The approved storage period shall apply. 2.2.6 Derivatization of polysaccharide
2.2.6.1. Dissolve the purified polysaccharide, and activate with cyanogens bromide in a proper ratio. Subsequently add adipic dihydrazide (ADH) in a appropriate concentration to the reaction
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solution to stay overnight. Then treat the reaction mixture with ultrafiltration or dialysis. The derivatized polysaccharide may be lyophilized to a solid state. 2.2.6.2. Control tests on the derivatized polysaccharide See section 3.2.
2.2.6.3. Storage and storage period
The derivatized polysaccharide shall be stored at or below -20℃. The storage period shall be not more than 30 days. 2.2.7 Carrier protein
Tetanus toxoid is used as the carrier protein. The production and quality control of the bulk tetanus toxoid shall comply with Sections 2.1-2.2 of the Tetanus Vaccine, Adsorbed.
The bulk tetanus toxoid can be further purified with column chromatography or other approved methods. It shall be diluted to an appropriate concentration. 2.2.8 Preparation of polysaccharide-protein conjugate 2.2.8.1 Conjugation
Mix equal amounts of the derivatized polysaccharide and tetanus toxoid, and add ethyl dimethylaminopropyl carbodiimide (EDAC) to start the reaction. Then treat the reaction mixture with ultrafiltration or dialysis. 2.2.8.2 Purification of conjugate
Purify the conjugate with column chromatography, collect the eluate around the void volume, then mix all the collected eluate from several times of column chromatography to make purified conjugate. After sterilization by filtration, the filtrate is regarded as the bulk conjugate. 2.2.9 Control tests on the bulk conjugate See Section 3.3
2.2.10 Storage and storage period
The bulk conjugate shall be stored at 2-8℃. The storage period shall be not more than 6 months. 2.3 Final bulk 2.3.1 Formulation
Dilute the bulk conjugate with sodium chloride injection. 1 ml of the vaccine solution shall contain not less than 20 μg of polysaccharide. 2.3.2 Control tests on the final bulk See Section 3.4 2.4 Final product 2.4.1 Defining batches
The Requirements for Defining Batches of Biologics shall apply. 2.4.2 Filling
The Requirements for Filling and Lyophilization of Biologics shall apply. 2.4.3 Specifications
0.5 ml per container. Each single human dose is 0.5 ml containing 10 μg of polysaccharide. 2.4.4 Packaging
The Requirements for Packaging of Biologics shall apply.
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