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A kind of preparation method of Haemophilus influenzae type b polysaccharide conjugate vaccine
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A Haemophilus influenzae and conjugate vaccine technology, which is applied in the field of preparation of Haemophilus influenzae type B polysaccharide conjugate vaccine, can solve the problems of unstable quality, complicated process and high cost
Active Publication Date: 2016-08-03
TASLY BIOPHARMACEUTICALS CO LTD
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[0005] The existing Hib conjugate vaccine production technology mainly includes the selection and cultivation of strains, the extraction, separation and purification of capsular polysaccharide, the cultivation, separation and purification of carrier protein, the combination of capsular polysaccharide and carrier protein, and the separation and purification of vaccine. , where the key steps have defects such as complex process, high cost, and unstable quality
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Embodiment 1
[0093] vaccine preparation
[0094] 1. Culture of Haemophilus influenzae type B;
[0095] The formula of the culture medium is that each liter of culture fluid contains the following components:
[0096]
[0097] The culture method is: shaking culture at 37 degrees Celsius and 150 rpm, after entering the logarithmic growth phase, adjust the pH to 7.4 with NaOH every 2 hours, co-cultivate for 24 hours, and sterilize with 0.6% formaldehyde after the culture.
[0098] 2. Extraction of polysaccharide extract of Haemophilus influenzae type B;
[0099] A, Extraction of PRP crude sugar: 1) Haemophilus influenzae type B culture solution 8000rpm10min to remove bacterial sediment, collect the supernatant; 2) pass the supernatant through a 0.22um hollow fiber, and harvest the filtrate; 3) Use the hollow fiber filtrate 50KDa membrane bag ultrafiltration concentration; 4) The membrane bag concentrated solution is subjected to graded alcohol precipitation: a. 25% low concentration alco...
Embodiment 2
[0104] Same as the method of Example 1, but in the cultivation of Haemophilus influenzae type B;
[0105] The formula of the culture medium is that each liter of culture fluid contains the following components:
[0106]
[0107] The culture method is: shake culture at 35°C, adjust the pH to 7 with NaOH every 1 hour after entering the logarithmic growth phase, co-cultivate for 24 hours, and sterilize after the culture is completed.
Embodiment 3
[0109] Same as the method of Example 1, but in the cultivation of Haemophilus influenzae type B;
[0110] The formula of the culture medium is that each liter of culture fluid contains the following components:
[0111]
[0112]
[0113] The culture method is: shake culture at 40°C, adjust the pH to 8 with NaOH every 3 hours after entering the logarithmic growth phase, co-cultivate for 24 hours, and sterilize after the culture is completed.
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Abstract
The invention discloses a preparation method of Haemophilus influenzae type B polysaccharide-conjugated vaccine. The preparation method is: extract the polysaccharide of Haemophilus influenzae type B, and dissolve PRP polysaccharide to 20 mg / ml with ultrapure water or water for injection at room temperature According to the condition that the mass ratio of PRP and CDAP is 1, CDAP is added and maintained for 1.5 min. Adjust the pH to 9.5 with 0.3M NaOH and maintain it for 3 minutes. Add solid ADH according to the condition that the mass ratio of ADH to PRP polysaccharide is 4:1. Adjust the pH to 9.5 with 0.3M NaOH and maintain it for 1h. 2-8°C, 0.2M NaCl dialysis; B. The dialyzed PRP-AH was mixed with 20mg / ml TT solution at a mass ratio of 1:2. Adjust the pH to 5.0 with 0.1M HCL, then add solid EDAC at a ratio of 10:1 mass ratio between EDAC and PRP, maintain the pH at room temperature at 5.060min, and adjust the pH to 7.5 with 0.3M NaOH for 30min.
Description
technical field [0001] The invention relates to a preparation method of a vaccine, in particular to a preparation method of a Haemophilus influenzae type B polysaccharide-conjugated vaccine. Background technique [0002] Haemophilus influenzae type b (Hib) is a highly invasive type of Haemophilus influenzae. Almost 90% of the serious infections caused by Haemophilus influenzae are caused by type B, mainly causing meningitis, Pneumonia, pericarditis, arthritis, bacteremia, epiglottitis and other diseases, the disease makes 15% to 35% of survivors suffer from permanent disability, typical symptoms include mental retardation or deafness. Meningitis and pneumonia caused by Haemophilus influenzae type b are the leading causes of infant mortality. [0003] Years of epidemiological studies have shown that the use of Hib vaccine is an effective measure to control Hib invasive disease. Therefore, it is necessary and important to develop a Hib vaccine with good effect and low cost. ...
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