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Freeze-drying method for producing diphtheria toxin non-toxic form CRM197 protein strain through fermentation
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A technology of CRM197 and diphtheria toxin, applied in the field of bioengineering, can solve the problem of low bacterial concentration and achieve the effects of low cost, short time consumption and simple freeze-drying curve
Active Publication Date: 2022-01-14
ZHEJIANG VACIN BIO PHARMA LTD
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Problems solved by technology
[0004] However, the lyophilized bacterial concentration of the current CRM197 protein strain lyophilized powder is low
Method used
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Embodiment 1
[0082] A strain freeze-drying method for fermenting and producing diphtheria toxin non-toxic form CRM197 protein, comprising the following steps:
[0083] S1, pre-freezing
[0084] Dilute the bacterial solution containing the lyoprotectant to a concentration of 1.0×10 6 , according to 0.5mL / cm 2 Lay flat on a freeze-dried stainless steel plate, place the freeze-dried stainless steel plate on a vacuum freeze-drying partition, and pre-freeze the conditions as follows: cold trap temperature ≤ -40°C, pre-freeze temperature -40°C, and maintain at normal pressure for 10h .
[0085] S2, sublimation drying
[0086] S2.1, first-order sublimation
[0087] The freeze-dried stainless steel plate is still placed on the partition plate of the vacuum freeze dryer, and the first-order sublimation conditions are: cold trap temperature ≤ -40°C, vacuum degree of 20pa, heating rate of 2°C / min, heating to -30°C, and maintaining 10h.
[0088] S2.2, second-order sublimation
[0089] The freez...
Embodiment 2-8
[0093] The difference from Example 1 is that the concentration of bacteria in the bacterial solution is different, see Table 7 for details.
[0095] Example Bacteria concentration Example 1 1.0×106 Example 2 5.0×106 Example 3 1.0×107 Example 4 5.0×107 Example 5 1.0×108 Example 6 5.0×108 Example 7 1.0×109 Example 8 5.0×109
Embodiment 9
[0097] A strain freeze-drying method for fermenting and producing diphtheria toxin non-toxic form CRM197 protein, comprising the following steps:
[0098] S1, pre-freezing
[0099] The bacteria solution containing the lyoprotectant was mixed at 0.1mL / cm 2 Lay flat on a freeze-dried stainless steel plate, place the freeze-dried stainless steel plate on a vacuum freeze-drying partition, and pre-freeze the conditions as follows: cold trap temperature ≤ -40°C, and maintain it for 2 hours, specifically, the concentration of the bacterial solution is 1 ×10 6 cfu / mL.
[0100] S2, sublimation drying
[0101] S2.1, first-order sublimation
[0102] The freeze-dried stainless steel plate is still placed on the partition of the vacuum freeze dryer, and the first-order sublimation conditions are: cold trap temperature ≤ -40°C, vacuum degree of 0 Pa, heating rate of 0.01°C / min, heating to -20°C, and maintaining 10h.
[0103] S2.2, second-order sublimation
[0104] The freeze-dried st...
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Abstract
The invention relates to the field of bioengineering, in particular to a freeze-drying method for producing a diphtheriatoxin non-toxic form CRM197 protein strain through fermentation. The method comprises the following steps: Step 1, spreading bacterial liquid containing a freeze-drying protective agent in a freeze-drying stainless steel plate, and placing the freeze-drying stainless steel plate on a vacuum freeze-drying partition plate; Step 2, performing sublimation drying; Step 2.1, performing first-stage sublimation, and still placing the freeze-drying stainless steel plate on a partition plate of a vacuum freeze-drying machine; Step 2.2, performing second-stage sublimation, and still placing the freeze-drying stainless steel plate on the partition plate of the vacuum freeze-drying machine; and Step 3, performing desorption drying, and still placing the freeze-drying stainless steel plate on a partition plate of the vacuum freeze-drying machine to prepare CRM197 strain freeze-dried powder. The effect of improving the bacterial concentration of the CRM197 strain freeze-dried powder after freeze-drying is achieved.
Description
technical field [0001] The present application relates to the field of bioengineering, more specifically, to a freeze-drying method for fermenting and producing a non-toxic CRM197 protein strain of diphtheriatoxin. Background technique [0002] CRM197 is a non-toxic diphtheriatoxinmutant with a single mutation replacing glycine at position 52 with glutamic acid (documents 1, 2), which changes the active site of diphtheria toxinenzyme, so that it cannot produce toxic effects on cells. However, it is still consistent with the natural diphtheria toxin in terms of antigenicity and immunogenicity. CRM197 is a well-studied protein that acts as a carrier of polysaccharides and haptens to render them immunogenic. It is used as a carrier protein in many approved vaccines, such as conjugate vaccines for meningococcal, Haemophilus influenzae type b and pneumococcal infections. [0003] Based on the multiple advantages of CRM197 protein, more and more companies use the non-toxic f...
Claims
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