Kit for removing bacterial endotoxin in biological product, method thereof, and preparation method of biological product

A technology of bacterial endotoxin and biological products, which is applied in the preparation method of peptides, biochemical equipment and methods, and the determination/inspection of microorganisms, etc. It can solve problems such as inability to separate, loss of active substances in plasmids, difficult industrial operation and control, etc. , to achieve the effect of low cost, less active material loss and easy operation

Active Publication Date: 2015-02-25
陈辉
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Common removal methods include ultrafiltration, various column chromatography (such as hydrophobic chromatography, ion exchange), affinity adsorption (polymyxin B, L-histidine, poly-L-lysine, poly-L-lysine, Poly-γ-L-glutamic acid cross-linking medium) and cloud point extraction (Triton X-114), etc., these methods can remove or inactivate part of endotoxin, but there are certain shortcomings
For example, ultrafiltration can only be applied to small-molecule drugs. If the drug molecules (such as antibodies) and endotoxin molecules are close in size, they cannot be effectively separated; column chromatography is costly and inefficient, and is not suitable for large-scale production of bacterial endotoxins. removal of the same negatively charged biomolecules by anion exchange resins; cloud point extraction (Triton X-114) [4] There are also great disadvantages such as the need for multiple extractions during the operation, resulting in the loss of active substances in the plasmid; the temperature needs to be switched during processing, and after the phase transition, Triton X-114 forms very fine droplets and exists in the water phase, which requires high-speed centrifugation , difficult to industrial operation and control, etc.

Method used

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  • Kit for removing bacterial endotoxin in biological product, method thereof, and preparation method of biological product
  • Kit for removing bacterial endotoxin in biological product, method thereof, and preparation method of biological product
  • Kit for removing bacterial endotoxin in biological product, method thereof, and preparation method of biological product

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0022] Experimental example 1, preparation work before removing endotoxin from biological product samples

[0023] 1. Drawing of endotoxin standard substance content-absorbance standard curve

[0024] 1. Experimental materials

[0025] The chromogenic substrate Limulus kit was purchased from Xiamen Limulus Reagent Experimental Factory Co., Ltd.; the experimental equipment used was free of endotoxin.

[0026] 2. Preparation of standard endotoxin solution and drawing of standard curve

[0027] All were carried out in accordance with the instruction manual of the chromogenic substrate LAL kit provided by Xiamen Limulus Reagent Experimental Factory Co., Ltd.

[0028] Measure the absorbance of the endotoxin standard substance by photometry (see Table 1), and take the absorbance as the Y axis and the endotoxin concentration as the X axis to draw the endotoxin concentration-absorbance standard curve (see Table 1). figure 1 ), obtain the standard curve equation of y=0.791X+0.084 (R...

Embodiment 1

[0046] Example 1. Removal of endotoxin in samples of endotoxin-removing biological products

[0047] 1) Take the endotoxin-removing biological product sample obtained by the method in the third part of Experimental Example 1, and add an anionic surfactant to it until the final concentration of the anionic surfactant is not less than 1 wt% (the concentration of the anionic surfactant depends on the endotoxin Concentration, the higher the concentration of endotoxin, the concentration of anionic surfactant also needs to increase correspondingly, generally around 5-10wt%), after mixing evenly, let it stand for 5min; the anionic surfactant can be sodium dodecyl sulfate (SDS), Sodium deoxycholate (SDC), sodium secondary alkyl sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, sodium oleyl alcohol sulfate, sodium N-oleoyl polypeptide, sodium alkylbenzenesulfonate, α-olefin sulfonic acid Sodium, sodium alkyl sulfonate, alpha-sulfomonocarboxylate, fatty acid sulfoalkyl ester,...

experiment example 2

[0051] Experimental example 2, endotoxin detection interference test

[0052] In order to exclude the influence of the absorbance of biological products on the absorbance of endotoxin, an interference test was carried out to screen out the concentration of biological products with the least degree of interference to the detection of endotoxin. The operation process of the interference test was carried out in accordance with the "interference test of the test product" in the instruction manual of the chromogenic substrate LAL kit provided by Xiamen Limulus Reagent Experimental Factory Co., Ltd.

[0053] 1. Interference test of protein sample solution on endotoxin detection

[0054] Since the protein sample solution is added with a certain concentration of other substances when the endotoxin is removed, it needs to be diluted to a certain multiple before determination to eliminate interference.

[0055] The endotoxin-removing protein sample solution (pH7-8) obtained in step 3) ...

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Abstract

The invention discloses a kit for removing bacterial endotoxin in a biological product, a method for removing the bacterial endotoxin in the biological product by using the kit, and a preparation method of the endotoxin removed biological product. The kit includes an anionic surfactant and a potassium salt. The methods commonly comprise the following steps: fully combining the anionic surfactant with the endotoxin in the biological product to form a conjugate, adding the potassium salt to precipitate the conjugate, filtering to remove the obtained precipitate in order to obtain an endotoxin removed biological product solution, and separating the biological product from the biological product solution. The kit can be used to simply and efficiently remove the endotoxin in the biological product and has a low cost, and the endotoxin removal method has the advantages of easy operation, no influences on the bioactivity of the biological product, and only use of compounds allowed in the medicine industry. The content of the endotoxin in the biological product prepared by using the methods accords with clinic pharmacy standards, and the loss of active substances is less.

Description

technical field [0001] The invention relates to the field of separation and purification of biological products, in particular to a kit for removing bacterial endotoxin from biological products, a removal method and a preparation method of biological products for removing endotoxin. Background technique [0002] With the wide application of modern biotechnology, more and more attention has been paid to the safety of biological products, especially the stricter control of pyrogens in biological products. However, in the production process of biological products, pyrogens are often introduced due to factors such as raw materials and auxiliary materials, production environment, and personal operation. Release active substances and introduce a large number of pyrogens, contaminating biological products. Therefore, in addition to taking effective process control and strict preventive measures in the production process to reduce the source of pyrogens, how to remove pyrogens in b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14C07K14/765C12N15/10
CPCC07K1/34C07K14/245C12N15/1017C12Q2527/125C07K1/30C07K14/765
Inventor 陈辉
Owner 陈辉
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