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A kind of freeze-drying method for producing diphtheria toxin non-toxic form crm197 protein strain by fermentation
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A technology of CRM197 and diphtheria toxin, applied in the field of bioengineering, can solve problems such as low bacterial concentration, and achieve the effects of low cost, simple freeze-drying curve and short time-consuming
Active Publication Date: 2022-08-05
艾美坚持生物制药有限公司
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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
[0084] A bacterial strain freeze-drying method for fermenting and producing a non-toxic form of diphtheria toxin CRM197 protein, comprising the following steps:
[0085] S1, pre-freezing
[0086] Dilute the bacterial solution containing the lyophilized protective agent to a concentration of 1.0×10 6 , according to 0.5mL / cm 2 Lay flat on a freeze-dried stainless steel plate, and place the freeze-dried stainless steel plate on a vacuum freeze-drying partition. The pre-freezing conditions are: cold trap temperature ≤ -40 °C, pre-freezing temperature -40 °C, and maintained at normal pressure for 10 hours .
[0087] S2, sublimation drying
[0088] S2.1, first-order sublimation
[0089] The freeze-dried stainless steel plate is still placed on the partition of the vacuum freeze-drying machine, and the first-order sublimation conditions are: cold trap temperature≤-40℃, vacuum degree 20pa, heating rate 2℃ / min, heating to -30℃, and maintaining 10h.
[0090] S2.2, second-order sub...
Embodiment 2-8
[0095] The difference from Example 1 is that the bacterial concentration of the bacterial solution is different, as shown in Table 7 for details.
[0096] Table 7 Bacterial concentration table
[0097] Example Bacterial concentration Example 1 1.0×10 6
Embodiment 9
[0099] A bacterial strain freeze-drying method for fermenting and producing a non-toxic form of diphtheria toxin CRM197 protein, comprising the following steps:
[0100] S1, pre-freezing
[0101] The bacterial solution containing the lyophilized protective agent was 0.1 mL / cm 2 Lay it on a freeze-dried stainless steel plate, place the freeze-dried stainless steel plate on a vacuum freeze-drying partition, and the pre-freezing conditions are: cold trap temperature ≤ -40 ° C, and maintain for 2 hours, specifically, the bacterial concentration is 1 ×10 6 cfu / mL.
[0102] S2, sublimation drying
[0103] S2.1, first-order sublimation
[0104] The freeze-dried stainless steel plate is still placed on the partition of the vacuum freeze-drying machine, and the first-order sublimation conditions are: cold trap temperature≤-40°C, vacuum degree 0pa, heating rate 0.01°C / min, heating to -20°C, and maintaining 10h.
[0105] S2.2, second-order sublimation
[0106] The freeze-dried sta...
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Abstract
The application relates to the field of bioengineering, and more specifically, to a lyophilization method for fermenting and producing diphtheria toxin non-toxic form CRM197 protein strains, comprising the following steps: S1, laying a bacterial liquid containing a lyophilized protective agent on a freezer In the dry stainless steel plate, the freeze-dried stainless steel plate is placed on the vacuum freeze-drying partition; S2, sublimation drying; S2.1, first-order sublimation, the freeze-dried stainless steel plate is still placed on the vacuum freeze-drying partition; S2 .2. Second-order sublimation, the freeze-dried stainless steel plate is still placed on the partition of the vacuum freeze-drying machine; S3, analytical drying, the freeze-dried stainless steel plate is still placed on the partition of the vacuum freeze-dried machine to obtain CRM197 strain freeze-dried powder ; The application has the effect of improving the bacterial concentration after freeze-drying of CRM197 strain freeze-dried powder.
Description
technical field [0001] The present application relates to the field of bioengineering, and more particularly, to a lyophilization method for producing CRM197 protein strains in a non-toxic form of diphtheria toxin by fermentation. Background technique [0002] CRM197 is a non-toxic diphtheria toxin mutant with a single mutation in which the 52-position glycine is replaced by glutamic acid (References 1, 2), resulting in changes in the active site of the diphtheria toxin enzyme, so that it cannot produce toxic effects on cells, However, the antigenicity and immunogenicity are still consistent with the natural diphtheria toxin. CRM197 is a well-studied protein that acts as a carrier for polysaccharides and haptens making it 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 various advantages of CRM197 protein, more and more ente...
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