CN102504042A

CN102504042A - Method for removing endotoxin from bacteria capsular polysaccharide

Info

Publication number: CN102504042A
Application number: CN2011103656502A
Authority: CN
Inventors: 吴强; 张丽莺; 李靖; 罗力心; 魏新; 赵丹; 韩炼; 陈庚; 关晓峰; 王子龙
Original assignee: Chengdu Olymvax Biopharmaceuticals Inc
Current assignee: Chengdu Olymvax Biopharmaceuticals Inc
Priority date: 2011-11-17
Filing date: 2011-11-17
Publication date: 2012-06-20

Abstract

The invention discloses a method for removing endotoxin from bacteria capsular polysaccharide, which comprises the following steps that: (1) TritonX-114 with the concentration of 8-12%v/v is added in bacteria capsular polysaccharide solution until the final concentration reaches 0.8-1.2%v/v; (2) the solution is mixed and kept at the temperature of 50-60 DEG C for 5-15 minutes; (3), after becoming layered, the solution is centrifuged in a device with the rotate speed of 3000-5000rpm, then supernate is collected, and primary extraction is completed; (4) the content of endotoxin in the supernate is detected by a sol-gel method, then extraction is repeated until the content of the endotoxin is less than 50EU/ug; and (5) the residual TritonX-114 is removed by dialysis. According to the method for removing endotoxin from bacteria capsular polysaccharide, the TritonX-114 is adopted, thereby achieving the advantages that the content of the endotoxin in the extracted bacteria capsular polysaccharide is reduced by 90% and the recovery of capsular polysaccharide is more than 85%, etc.

Description

Endotoxic method in a kind of removal bacterial capsule polysaccharide

Technical field

The present invention relates to endotoxic method in a kind of removal bacterial capsule polysaccharide.

Background technology

Bacteriotoxin can be divided into two types; One type is extracellular toxin (Exotoxin); It is a kind of toxic protein; Be that bacterium is secreted into the toxicant outside the thalline in process of growth, producing ectotoxic bacterium mainly is gram-positive microorganism, like diphtheria corynebacterium, tetanus bacillus, Clostridium botulinum, streptococcus aureus and minority Gram-negative bacteria; Another kind of is intracellular toxin (Endotoxin); It is a kind of LPS (the Lipoply Saccharide on the gram-negative bacteria cell wall; LPS) and the mixture of trace of albumin; Its singularity is not the meta-bolites of bacterium, but bacterial death or disintegrate after just discharge a kind ofly have a bioactive material of intracellular toxin, its Chemical Composition mainly is made up of O-specificity chain, core polysaccharide, lipoid A three parts.

Many bioengineering products are expressed by gram negative bacterium such as E.coli; After the bacteria breaking; Can contain a large amount of intracellular toxins in the sample, intracellular toxin has significant pyrogenicity property to mammal, when endotoxin content surpasses certain standard; Possibly cause side reactions such as more serious heating and anaphylactoid purpura, so each traditional Chinese medicines is examined all there is strictness in department to the endotoxin content in the biological products regulation.

Because the intracellular toxin of Gram-negative bacteria is a kind of LPS; Very similar with the bacterial capsule polysaccharide structures; Therefore from bacterial vaccine particularly Gram-negative bacteria be that to remove intracellular toxin the capsular polysaccharide vaccine product on basis is produced very difficult; At present; Commonly used separation of bacterial capsular polysaccharide and endotoxic method comprise affinity chromatography, active carbon adsorption, microporous membrane partition method, and these methods all are difficult to reach separation of bacterial capsular polysaccharide and endotoxic purpose, the endotoxic removal technology in the gram negative bacterium polysaccharide product of world health organisation recommendations be under the condition of 100000g cf-ultracentrifugation 4 by 6 hours; But this technology not only needs expensive superspeed refrigerated centrifuge, and the sample size of primary treatment is restricted.

Summary of the invention

The object of the invention promptly is to overcome the deficiency of prior art; A kind of wetting ability and endotoxic hydrophobicity of utilizing capsular polysaccharide is provided; Adopt TritonX-114 as extraction agent, the intracellular toxin in the b type hemophilus influenzae capsular polysaccharide solution is carried out the one or many extraction, remove the intracellular toxin in the capsular polysaccharide solution preferably; Adopt the TritonX-114 extraction process; Single extraction makes the endotoxin content in the b type hemophilus influenzae capsular polysaccharide solution descend 90%, and the capsular polysaccharide recovery is more than 85%, can reduce endotoxic method in a kind of removal bacterial capsule polysaccharide of endotoxin content in the bacterial capsule polysaccharide product effectively, easily.

The objective of the invention is to realize through following technical scheme: endotoxic method in a kind of removal bacterial capsule polysaccharide, it may further comprise the steps:

(1) in the bacterial capsule polysaccharide soln, adding the TritonX-114 that concentration is 8～12%v/v gradually, is 0.8～1.2%v/v up to the final concentration of mixing solutions, mixes 25～35 minutes;

(2) mix after, place under 50～60 ℃ of conditions, observe the layering situation after 5～15 minutes;

(3) treat the solution layering after, place and carry out centrifugally automatically in the automatic centrifugal device, automatically the rotating speed of centrifugal device is 3000～5000 rev/mins, centrifugation time is 8～12 minutes, collection supernatant centrifugal completions after, completion single extraction;

(4) detect in (3) endotoxin content in gained supernatant with the gel method TAL, repeat above extraction step, until endotoxin content less than 50 EU/ μ g;

(5) with gained supernatant in the pyrogen-free water for injection dialysis (4), dialysis time 20～28 hours, remaining TritonX-114 is removed in dialysis.

The invention has the beneficial effects as follows: the present invention provides endotoxic method in a kind of removal bacterial capsule polysaccharide; Utilize the wetting ability and the endotoxic hydrophobicity of capsular polysaccharide; Adopt TritonX-114 as extraction agent, the intracellular toxin in the b type hemophilus influenzae capsular polysaccharide solution is carried out the one or many extraction, remove the intracellular toxin in the capsular polysaccharide solution preferably; Adopt the TritonX-114 extraction process; Single extraction makes the endotoxin content in the b type hemophilus influenzae capsular polysaccharide solution descend 90%, and the capsular polysaccharide recovery can reduce endotoxin content in the bacterial capsule polysaccharide product effectively, easily more than 85%.

Embodiment

Below in conjunction with embodiment the present invention is done further description, but protection scope of the present invention is not limited to the following stated.

Embodiment 1:

Endotoxic method in a kind of removal bacterial capsule polysaccharide, it may further comprise the steps:

(1) in the bacterial capsule polysaccharide soln, adding the TritonX-114 that concentration is 8%v/v gradually, is 1.2%v/v up to the final concentration of mixing solutions, mixes 35 minutes;

(2) mix after, place under 50 ℃ of conditions, observe the layering situation after 15 minutes;

(3) treat the solution layering after, place and carry out centrifugally automatically in the automatic centrifugal device, automatically the rotating speed of centrifugal device is 3000 rev/mins, centrifugation time is 12 minutes, collection supernatant centrifugal completions after, completion single extraction;

(5) with gained supernatant in the pyrogen-free water for injection dialysis (4), dialysis time 20 hours, remaining TritonX-114 is removed in dialysis.

Embodiment 2:

(1) in the bacterial capsule polysaccharide soln, adding the TritonX-114 that concentration is 12%v/v gradually, is 0.8%v/v up to the final concentration of mixing solutions, mixes 25 minutes;

(2) mix after, place under 60 ℃ of conditions, observe the layering situation after 5 minutes;

(3) treat the solution layering after, place and carry out centrifugally automatically in the automatic centrifugal device, automatically the rotating speed of centrifugal device is 5000 rev/mins, centrifugation time is 8 minutes, collection supernatant centrifugal completions after, completion single extraction;

(5) with gained supernatant in the pyrogen-free water for injection dialysis (4), dialysis time 28 hours, remaining TritonX-114 is removed in dialysis.

Embodiment 3:

(1) in the bacterial capsule polysaccharide soln, adding the TritonX-114 that concentration is 10%v/v gradually, is 1.0%v/v up to the final concentration of mixing solutions, mixes 30 minutes;

(2) mix after, place under 55 ℃ of conditions, observe the layering situation after 10 minutes;

(3) treat the solution layering after, place and carry out centrifugally automatically in the automatic centrifugal device, automatically the rotating speed of centrifugal device is 4000 rev/mins, centrifugation time is 10 minutes, collection supernatant centrifugal completions after, completion single extraction;

(5) with gained supernatant in the pyrogen-free water for injection dialysis (4), dialysis time 24 hours, remaining TritonX-114 is removed in dialysis.

Claims

1. remove endotoxic method in the bacterial capsule polysaccharide for one kind, it is characterized in that: it may further comprise the steps:

(4) detect in (3) endotoxin content in gained supernatant with gel method, repeat above extraction step, until endotoxin content less than 50 EU/ μ g;