CN1970780A - Process for removing endotoxin in bacteria polysaccharide by using macroporous resin - Google Patents
Process for removing endotoxin in bacteria polysaccharide by using macroporous resin Download PDFInfo
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- CN1970780A CN1970780A CN 200610048878 CN200610048878A CN1970780A CN 1970780 A CN1970780 A CN 1970780A CN 200610048878 CN200610048878 CN 200610048878 CN 200610048878 A CN200610048878 A CN 200610048878A CN 1970780 A CN1970780 A CN 1970780A
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Abstract
The invention discloses a removing method of endotoxin in the bacterial polysaccharide through large-hole resin in the biological technical domain, which comprises the following steps: fermenting bacteria through biological reactor; making rough sugar; extracting rough sugar through cooled phenol to remove protein; hyperfiltering through composite buffer of calcium ionic chelant-surface activator; removing endotoxin adsorbed by large-hole resin; obtaining bacterial polysaccharide with low endotoxin content.
Description
Technical field:
The invention belongs to biological technical field, more specifically, relate to endotoxic removal method in the bacterial polysaccharides.
Background technology:
The bacterial capsule polysaccharide is the important protective antigen of bacterium, inoculates these polysaccharide vaccines, can make the colony more than 2 years old obtain immunoprotection.But in gram negative bacterium fermentation culture process, except producing capsular polysaccharide, compositions such as also synthetic a large amount of protein, nucleic acid, intracellular toxin.When endotoxin in vaccine content surpasses certain standard, may cause side reactions such as more serious heating and anaphylactoid purpura after the inoculation, so the bacterial polysaccharides vaccine rules of The World Health Organization (WHO) issue and " standard of 2005 editions three middle polysaccharide vaccines of Chinese pharmacopoeia all has strict requirement to endotoxin content in the capsular polysaccharide.
The intracellular toxin that gram negative bacterium produces is not easy to remove in the polysaccharide purification process.The endotoxic removal technology in Gram-negative bacterioid polysaccharide product that The World Health Organization (WHO) is recommended is for adopting 100, and ultracentrifugation is 4～6 hours under the centrifugal force condition of 000g.But this technology not only needs the very expensive superspeed refrigerated centrifuge of price, and the sample size of primary treatment is restricted, for the large-scale production of vaccine brings difficulty.
Adopt affinity chromatography to remove intracellular toxin in the bacterial polysaccharides, go the intracellular toxin affinity gel to endotoxic loading capacity generally at 3000-7000 EU/ml, and the endotoxin content in the Gram-negative bacterioid polysaccharide product is generally in 1-2 * 10
6The level of EU/mg is so affinity gel is removed the restriction that endotoxic method is subjected to certain condition.
Macroporous resin is the resin of a class chemosynthesis, has the high characteristics of chemical stability, the existing extensive application in antibiotic purification and phenol recovery.But being used for Gram-negative bacterioid polysaccharide product endotoxin removal, macroporous resin still do not have bibliographical information at present both at home and abroad.
Summary of the invention:
The present invention has overcome the defective and the deficiency of prior art, and a kind of efficient, convenient, method of being easy to amplify that obtains the low bacterial polysaccharides of endotoxin content is provided.
Technical scheme of the present invention and step:
(1) adopt bio-reactor that bacterium is carried out fermentation culture;
(2) after the sterilization of logarithmic growth later stage, centrifugal collection supernatant liquor;
(3) in supernatant liquor, add cetyl trimethylammonium bromide, centrifugal collecting precipitation;
(4) add 1mol/L CaCl in the precipitation
2Solution dissociates, and adds ethanol to final concentration 25%, centrifugal collection supernatant liquor;
(5) supernatant liquor adds ethanol to 50～90%; Centrifugal collection polysaccharide precipitation;
(6) polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively;
(7) polysaccharide after the washing obtains rough polysaccharide through lyophilize;
(8) raw sugar is dissolved in the 1/10 saturated neutral sodium acetate soln by 5～50mg/ml, with cold phenol extraction 1～10 time, and centrifugal collection supernatant liquor;
(9) supernatant liquor adopts the cocktail buffer ultrafiltration that contains 1～15% calcium ion chelator and 0.1～2.0% tensio-active agent;
(10) in ultrafiltration and concentration liquid, add pretreated macroporous resin, adsorbed 4～8 hours;
Under the condition of (11) 2000～5000rpm centrifugal 10～60 minutes, collect supernatant liquor;
(12) supernatant liquor adopts water for injection, and 3～300KD film carries out ultrafiltration;
(13) add CaCl in the ultrafiltration and concentration liquid
2Solution is to final concentration 0.1-2mol/L, and alcohol concn 20～30% precipitates, centrifugal collection supernatant liquor;
(14) supernatant liquor adopts 3～300KD film to carry out ultrafiltration;
(15) liquid adds CaCl after the ultrafiltration
2Or NaCl solution is to final concentration 0.1-2mol/L, and ethanolic soln is to final concentration 50-90% precipitation polysaccharide, centrifugal collection polysaccharide precipitation;
(16) polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively;
(17) Xi Di polysaccharide obtains the low refining polysaccharide of endotoxin content through lyophilize.
Wherein bacterium can for following any: b type hemophilus influenzae, A group meningitis cocci, C group meningitis cocci, W
135Group meningitis cocci, Y group meningitis cocci, salmonella typhi; Macroporous resin is for having porous polystyrene-divinylbenzene pedestal, can for following any: D3520, X-5, HP20; The raw sugar concentration of ordinary dissolution can be 30～50mg/ml, cold phenol extraction 6～10 times; Calcium ion chelator can for following any: sodium ethylene diamine tetracetate, trisodium citrate, concentration are 8～15% (W/V); Tensio-active agent can for following any: TritonX-100, Tween-20, Sodium desoxycholate, concentration are 1.2～2.0% (W/V); Used ultra-filtration membrane molecular weight cut-off can be 100～300KD; The polysaccharide that is obtained is fit to preparation bacterial polysaccharides vaccine and polysaccharide protein combined vaccine.
The invention has the beneficial effects as follows on the basis of the characteristic of fully understanding macroporous resin and action principle, initiative is applied to endotoxic removal in the Gram-negative bacterioid polysaccharide product with macroporous resin, by the macroporous resin absorption effect, can be with the lipopolysaccharides absorption that has than strong-hydrophobicity, keep bacterial polysaccharides, thereby obtained to be suitable for preparing the low bacterial polysaccharides of endotoxin content of vaccine.Therefore, utilize macroporous resin to remove endotoxic method and substitute existing endotoxin removal technology, make that the endotoxin removal effect of bacterial polysaccharides is better than the WHO recommend method, realized the new breakthrough on bacterial polysaccharides endotoxin removal method for many years.
Embodiment:
Embodiment 1
Adopt bio-reactor that b type hemophilus influenzae is carried out fermentation culture; After the usefulness formaldehyde sterilization of logarithmic growth later stage, centrifugal collection supernatant liquor; Add cetyl trimethylammonium bromide to final concentration 0.1% in supernatant liquor, room temperature left standstill 12 hours, centrifugal collecting precipitation; Add 1mol/L CaCl in the precipitation
2Solution stirred after 3 hours, added ethanol to final concentration 25%, centrifugal collection supernatant liquor; Supernatant liquor adds ethanol to final concentration 50%, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation is used dehydrated alcohol, each washed twice of acetone respectively; Polysaccharide after the washing obtains the rough polysaccharide of b type hemophilus influenzae through vacuum lyophilization.
The 50g raw sugar is dissolved in the saturated neutral sodium acetate soln of 1/10 (V/V) by 5mg/ml, with cold phenol extraction 1 time, centrifugal collection supernatant liquor.
Supernatant adopts the 3KD ultra-filtration membrane with 20 times of volume 1% sodium ethylene diamine tetracetates-0.1%Triton X-100 damping fluid ultrafiltration; In ultrafiltration and concentration liquid, add pretreated macroporous resin D-3520, adsorbed 4 hours.
After absorption is finished, under 8 ℃, the condition of 2000rpm centrifugal 10 minutes, collect supernatant liquor; Supernatant liquor adopts the 3KD ultra-filtration membrane with the ultrafiltration of 30 times of volume water for injection, removes sodium ethylene diamine tetracetate and Triton X-100; Add CaCl in the ultrafiltration and concentration liquid
2Solution is to final concentration 0.1mol/L, and alcohol concn 20% precipitates, and centrifugal 1 hour of 10000rpm, supernatant liquor adopt the 3KD film to carry out ultrafiltration; Liquid adds sodium chloride solution to final concentration 0.1mol/L after the ultrafiltration, and ethanolic soln is to final concentration 50% precipitation polysaccharide, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively; The polysaccharide of washing is done through freezing, obtains the refining b type hemophilus influenzae polysaccharide of 3.47g.
Every calibrating index is as follows:
Protein content: 0.31%
Nucleic acid content: 0.42%
Ribose content: 38.2%
Phosphorus content: 7.6%
K
DMolecule less than 0.5 reclaims: 78.5%
Telling test: produce precipitation line with b type hemophilus influenzae specific corrosioning anteserum
Endotoxin content: 4EU/ microgram polysaccharide
Embodiment 2
Adopt bio-reactor that the A group meningitis cocci is carried out fermentation culture; After the usefulness formaldehyde sterilization of logarithmic growth later stage, centrifugal collection supernatant liquor; Add cetyl trimethylammonium bromide to final concentration 0.1% in supernatant liquor, room temperature left standstill 12 hours, centrifugal collecting precipitation; Add 1mol/LCaCl in the precipitation
2Solution stirred after 3 hours, added ethanol to final concentration 25%, centrifugal collection supernatant liquor; Supernatant liquor adds ethanol to final concentration 90%, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively; Polysaccharide after the washing obtains the rough polysaccharide of A group meningitis cocci through vacuum lyophilization.
The 500g raw sugar is dissolved in the saturated neutral sodium acetate soln of 1/10 (V/V) by 50mg/ml, with cold phenol extraction 10 times, centrifugal collection supernatant liquor.
Supernatant liquor adopts the cocktail buffer ultrafiltration of 300KD ultra-filtration membrane with 20 times of volume 15% sodium ethylene diamine tetracetates-2%Triton X-100; In ultrafiltration and concentration liquid, add pretreated macroporous resin X-5, adsorbed 8 hours.
After absorption is finished, under 8 ℃, the condition of 5000rpm centrifugal 60 minutes, collect supernatant liquor; Supernatant liquor adopts the 300KD ultra-filtration membrane with the ultrafiltration of 30 times of volume water for injection, removes sodium ethylene diamine tetracetate and Triton X-100; Add CaCl in the ultrafiltration and concentration liquid
2Solution is to final concentration 2mol/L, and alcohol concn 30% precipitates, and centrifugal 1 hour of 10000rpm, supernatant liquor adopt the 300KD film to carry out ultrafiltration; Liquid adds sodium-chlor to final concentration 2mol/L after the ultrafiltration, and ethanolic soln is to final concentration 90% precipitation polysaccharide, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively; The polysaccharide of washing obtains the refining A group meningitis cocci polysaccharide of 105g through lyophilize.
Every calibrating index is as follows:
Protein content: 0.22%
Nucleic acid content: 0.31%
O-acetyl content: 2.3mmol/g
Phosphorus content: 85mg/g
K
DMolecule less than 0.5 reclaims: 91.2%
Telling test: produce precipitation line with A group meningitis cocci specific corrosioning anteserum
Endotoxin content: 1EU/ microgram polysaccharide
Embodiment 3
Adopt bio-reactor that the C group meningitis cocci is carried out fermentation culture; After the usefulness formaldehyde sterilization of logarithmic growth later stage, centrifugal collection supernatant liquor; Add cetyl trimethylammonium bromide to final concentration 0.1% in supernatant liquor, room temperature left standstill 12 hours, centrifugal collecting precipitation; Add 1mol/LCaCl in the precipitation
2Solution stirred after 3 hours, added ethanol to final concentration 25%, centrifugal collection supernatant liquor; Supernatant liquor adds ethanol to final concentration 80%, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively; Polysaccharide after the washing obtains the rough polysaccharide of C group meningitis cocci through vacuum lyophilization.
The 500g raw sugar is dissolved in the saturated neutral sodium acetate soln of 1/10 (V/V) by 30mg/ml, with cold phenol extraction 6 times, centrifugal collection supernatant liquor.
Supernatant liquor adopts the cocktail buffer ultrafiltration of 100KD ultra-filtration membrane with 20 times of volume 8% sodium ethylene diamine tetracetate-1.2% Triton X-100; In ultrafiltration and concentration liquid, add pretreated macroporous resin HP20, adsorbed 6 hours.
After absorption is finished, under 8 ℃, the condition of 4000rpm centrifugal 30 minutes, collect supernatant liquor; Supernatant liquor adopts the 100KD ultra-filtration membrane with the ultrafiltration of 30 times of volume water for injection, removes sodium ethylene diamine tetracetate and Triton X-100; Add CaCl in the ultrafiltration and concentration liquid
2Solution is to final concentration 0.5mol/L, and alcohol concn 25% precipitates, and centrifugal 1 hour of 10000rpm, supernatant liquor adopt the 100KD film to carry out ultrafiltration; Liquid adds calcium chloride to final concentration 0.1mol/L after the ultrafiltration, and ethanolic soln is to final concentration 75% precipitation polysaccharide, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively; The polysaccharide of washing obtains the refining C group meningitis cocci polysaccharide of 42.3g through lyophilize.
Every calibrating index is as follows:
Protein content: 0.24%
Nucleic acid content: 0.26%
O-acetyl content: 2.1mmol/g
Sialic acid content: 832mg/g
K
DMolecule less than 0.5 reclaims: 93.2%
Telling test: produce precipitation line with C group meningitis cocci specific corrosioning anteserum
Endotoxin content: 2EU/ microgram polysaccharide
Embodiment 4
Adopt bio-reactor to W
135Group meningitis cocci carries out fermentation culture; After the usefulness formaldehyde sterilization of logarithmic growth later stage, centrifugal collection supernatant liquor; Add cetyl trimethylammonium bromide to final concentration 0.1% in supernatant liquor, room temperature left standstill 12 hours, centrifugal collecting precipitation; Add 1mol/LCaCl in the precipitation
2Solution stirred after 3 hours, added ethanol to final concentration 25%, centrifugal collection supernatant liquor; Supernatant liquor adds ethanol to final concentration 75%, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively; Polysaccharide after the washing obtains W through vacuum lyophilization
135The rough polysaccharide of group meningitis cocci.
The 50g raw sugar is dissolved in the saturated neutral sodium acetate soln of 1/10 (V/V) by 20mg/ml, with cold phenol extraction 5 times, centrifugal collection supernatant liquor.
Supernatant liquor adopts the cocktail buffer ultrafiltration of 10KD ultra-filtration membrane with 20 times of volume 1% trisodium citrate-0.1% Tween-20; In ultrafiltration and concentration liquid, add pretreated macroporous resin D-3520, adsorbed 5 hours.
After absorption is finished, under 8 ℃, the condition of 4000rpm centrifugal 30 minutes, collect supernatant liquor; Supernatant liquor adopts the 10KD ultra-filtration membrane with the ultrafiltration of 30 times of volume water for injection, removes trisodium citrate and Tween-20; Add CaCl in the ultrafiltration and concentration liquid
2Solution is to final concentration 0.3mol/L, and alcohol concn 27% precipitates, and centrifugal 1 hour of 10000rpm, supernatant liquor adopt the 10KD film to carry out ultrafiltration; Liquid adds calcium chloride to final concentration 2mol/L after the ultrafiltration, and ethanolic soln is to final concentration 65% precipitation polysaccharide, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively; The polysaccharide of washing obtains the refining W of 3.52g through lyophilize
135The group meningitis cocci polysaccharide.
Every calibrating index is as follows:
Protein content: 0.31%
Nucleic acid content: 0.35%
O-acetyl content: 0.58mmol/g
Sialic acid content: 615mg/g
K
DMolecule less than 0.5 reclaims: 90.2%
Telling test: with W
135The group meningitis cocci specific corrosioning anteserum produces precipitation line
Endotoxin content: 10EU/ microgram polysaccharide
Embodiment 5
Adopt bio-reactor that the Y group meningitis cocci is carried out fermentation culture; After the usefulness formaldehyde sterilization of logarithmic growth later stage, centrifugal collection supernatant liquor; Add cetyl trimethylammonium bromide to final concentration 0.1% in supernatant liquor, room temperature left standstill 12 hours; Centrifugal collecting precipitation adds 1mol/L CaCl in the precipitation
2Solution dissociates, and stirs after 3 hours, adds ethanol to final concentration 25%, centrifugal collection supernatant liquor; Supernatant liquor adds ethanol to final concentration 80%, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively; Polysaccharide after the washing obtains the rough polysaccharide of Y group meningitis cocci through vacuum lyophilization.
The 45g raw sugar is dissolved in the 1/10 saturated neutral sodium acetate soln by 10mg/ml, with cold phenol extraction 4 times, centrifugal collection supernatant liquor.
Supernatant liquor adopts 15% trisodium citrate-2% Tween-20 of 30kD ultra-filtration membrane with 20 times of volumes; In ultrafiltration and concentration liquid, add pretreated macroporous resin HP20, adsorbed 7 hours;
After absorption is finished, under 8 ℃, the condition of 2500rpm centrifugal 25 minutes, collect supernatant liquor; Supernatant liquor adopts the 30KD ultra-filtration membrane with the ultrafiltration of 30 times of volume water for injection, removes trisodium citrate and Tween-20; Add CaCl in the ultrafiltration and concentration liquid
2Solution is to final concentration 1.0mol/L, and alcohol concn 26% precipitates, centrifugal collection supernatant liquor; Supernatant liquor adopts the 30KD film to carry out ultrafiltration; Liquid adds sodium chloride solution to final concentration 1.0mol/L after the ultrafiltration, and ethanolic soln is to final concentration 60% precipitation polysaccharide, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively; The polysaccharide of washing obtains the refining Y group meningitis cocci polysaccharide of 3.78g through lyophilize.
Every calibrating index is as follows:
Protein content: 0.42%
Nucleic acid content: 0.39%
O-acetyl content: 0.62mmol/g
Sialic acid content: 596mg/g
K
DMolecule less than 0.5 reclaims: 93.5%
Telling test: produce precipitation line with Y group meningitis cocci specific corrosioning anteserum
Endotoxin content: 8EU/ microgram polysaccharide
Embodiment 6
Adopt bio-reactor that salmonella typhi is carried out fermentation culture; After the usefulness formaldehyde sterilization of logarithmic growth later stage, centrifugal collection supernatant liquor; Add cetyl trimethylammonium bromide to final concentration 0.1% in supernatant liquor, room temperature left standstill 12 hours, centrifugal collecting precipitation; Add 1mol/L CaCl in the precipitation
2Solution stirred after 3 hours, added ethanol to final concentration 25%, centrifugal collection supernatant liquor; Supernatant liquor adds ethanol to final concentration 75%, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation is used dehydrated alcohol, each washed twice of acetone respectively; Polysaccharide after the washing obtains the rough polysaccharide of typhoid fever through vacuum lyophilization.
The 15g raw sugar is dissolved in the saturated neutral sodium acetate soln of 1/10 (V/V) by 5mg/ml, with cold phenol extraction 3 times, centrifugal collection supernatant liquor.
Supernatant liquor adopts the 50KD ultra-filtration membrane with the ultrafiltration of 20 times of volume 8% trisodium citrate-1.2% Tween-20 damping fluids; In ultrafiltration and concentration liquid, add pretreated macroporous resin X-5, adsorbed 4 hours.
After absorption is finished, under 8 ℃, the condition of 2000rpm centrifugal 10 minutes, collect supernatant liquor; Supernatant liquor adopts the 50KD ultra-filtration membrane with the ultrafiltration of 30 times of volume water for injection, removes trisodium citrate and Tween-20; Add CaCl in the ultrafiltration and concentration liquid
2Solution is to final concentration 0.5mol/L, and alcohol concn 25% precipitates, and centrifugal 1 hour of 10000rpm, supernatant liquor adopt the 50KD film to carry out ultrafiltration; Liquid adds calcium chloride to 0.3mol/L after the ultrafiltration, and ethanolic soln is to final concentration 75% precipitation polysaccharide, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively; The polysaccharide of washing obtains the refining typhoid Vi polysaccharide of 1650mg through lyophilize.
Every calibrating index is as follows:
Protein content: 0.34%
Nucleic acid content: 0.28%
O-acetyl content: 2.86mmol/g
K
DMolecule less than 0.25 reclaims: 67.4%
Telling test: form precipitation line with the special serum of Salmonella typhoid Vi, with typhoid fever O serum not
Form precipitation line
Endotoxin content: 4EU/ microgram polysaccharide
Embodiment 7
Adopt bio-reactor to W
135Group meningitis cocci carries out fermentation culture; After the usefulness formaldehyde sterilization of logarithmic growth later stage, centrifugal collection supernatant liquor; Add cetyl trimethylammonium bromide to final concentration 0.1% in supernatant liquor, room temperature left standstill 12 hours, centrifugal collecting precipitation; Add 1mol/LCaCl in the precipitation
2Solution stirred after 3 hours, added ethanol to final concentration 25%, centrifugal collection supernatant liquor; Supernatant liquor adds ethanol to final concentration 75%, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively; Polysaccharide after the washing obtains W through vacuum lyophilization
135The rough polysaccharide of group meningitis cocci.
The 100g raw sugar is dissolved in the saturated neutral sodium acetate soln of 1/10 (V/V) by 25mg/ml, with cold phenol extraction 5 times, centrifugal collection supernatant liquor.
Supernatant liquor adopts the cocktail buffer ultrafiltration of 100KD ultra-filtration membrane with 20 times of volume 8% trisodium citrate-0.1% Sodium desoxycholates; In ultrafiltration and concentration liquid, add pretreated macropore tree D-3520, adsorbed 5 hours.
After absorption is finished, under 8 ℃, the condition of 4000rpm centrifugal 30 minutes, collect supernatant liquor; Supernatant liquor adopts the 100KD ultra-filtration membrane with the ultrafiltration of 30 times of volume water for injection, removes trisodium citrate and Sodium desoxycholate; Add CaCl in the ultrafiltration and concentration liquid
2Solution is to final concentration 0.3mol/L, and alcohol concn 28% precipitates, and centrifugal 1 hour of 10000rpm, supernatant liquor adopt the 100KD film to carry out ultrafiltration; Liquid adds calcium chloride to final concentration 0.5mol/L after the ultrafiltration, and ethanolic soln is to final concentration 75% precipitation polysaccharide, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively; The polysaccharide of washing obtains the refining W of 8.52g through lyophilize
135The group meningitis cocci polysaccharide.
Every calibrating index is as follows:
Protein content: 0.21%
Nucleic acid content: 0.25%
O-acetyl content: 0.56mmol/g
Sialic acid content: 610mg/g
K
DMolecule less than 0.5 reclaims: 95.2%
Telling test: with W
135The group meningitis cocci specific corrosioning anteserum produces precipitation line
Endotoxin content: 8EU/ microgram polysaccharide
Embodiment 8
Adopt bio-reactor that the Y group meningitis cocci is carried out fermentation culture; After the usefulness formaldehyde sterilization of logarithmic growth later stage, centrifugal collection supernatant liquor; Add cetyl trimethylammonium bromide to final concentration 0.1% in supernatant liquor, room temperature left standstill 12 hours; Centrifugal collecting precipitation adds 1mol/L CaCl in the precipitation
2Solution dissociates, and stirs after 3 hours, adds ethanol to final concentration 25%, centrifugal collection supernatant liquor; Supernatant liquor adds ethanol to final concentration 80%, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively; Polysaccharide after the washing obtains the rough polysaccharide of Y group meningitis cocci through vacuum lyophilization.
The 60g raw sugar is dissolved in the 1/10 saturated neutral sodium acetate soln by 10mg/ml, with cold phenol extraction 3 times, centrifugal collection supernatant liquor.
Supernatant liquor adopts 9% trisodium citrate-2% Sodium desoxycholate of 50kD ultra-filtration membrane with 20 times of volumes; In ultrafiltration and concentration liquid, add pretreated macroporous resin HP20, adsorbed 4 hours;
After absorption is finished, under 8 ℃, the condition of 2500rpm centrifugal 25 minutes, collect supernatant liquor; Supernatant liquor adopts the 50KD ultra-filtration membrane with the ultrafiltration of 30 times of volume water for injection, removes trisodium citrate and Sodium desoxycholate; Add CaCl in the ultrafiltration and concentration liquid
2Solution is to final concentration 1.0mol/L, and alcohol concn 26% precipitates, centrifugal collection supernatant liquor; Supernatant liquor adopts the 50KD film to carry out ultrafiltration; Liquid adds sodium chloride solution to final concentration 0.3mol/L after the ultrafiltration, and ethanolic soln is to final concentration 70% precipitation polysaccharide, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively; The polysaccharide of washing obtains the refining Y group meningitis cocci polysaccharide of 4.85g through lyophilize.
Every calibrating index is as follows:
Protein content: 0.32%
Nucleic acid content: 0.36%
O-acetyl content: 0.60mmol/g
Sialic acid content: 626mg/g
K
DMolecule less than 0.5 reclaims: 92.3%
Telling test: produce precipitation line with Y group meningitis cocci specific corrosioning anteserum
Endotoxin content: 8EU/ microgram polysaccharide
Embodiment 9
Adopt bio-reactor that the Y group meningitis cocci is carried out fermentation culture; After the usefulness formaldehyde sterilization of logarithmic growth later stage, centrifugal collection supernatant liquor; Add cetyl trimethylammonium bromide to final concentration 0.1% in supernatant liquor, room temperature left standstill 12 hours; Centrifugal collecting precipitation adds 1mol/L CaCl in the precipitation
2Solution dissociates, and stirs after 3 hours, adds ethanol to final concentration 25%, centrifugal collection supernatant liquor; Supernatant liquor adds ethanol to final concentration 80%, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively; Polysaccharide after the washing obtains the rough polysaccharide of Y group meningitis cocci through vacuum lyophilization.
The 40g raw sugar is dissolved in the 1/10 saturated neutral sodium acetate soln by 20mg/ml, with cold phenol extraction 3 times, centrifugal collection supernatant liquor.
Supernatant liquor adopts 10% trisodium citrate-1.2% Sodium desoxycholate of 100kD ultra-filtration membrane with 20 times of volumes; In ultrafiltration and concentration liquid, add pretreated macroporous resin X-5, adsorbed 4 hours;
After absorption is finished, under 8 ℃, the condition of 3500rpm centrifugal 30 minutes, collect supernatant liquor; Supernatant liquor adopts the 100KD ultra-filtration membrane with the ultrafiltration of 30 times of volume water for injection, removes trisodium citrate and Sodium desoxycholate; Add CaCl in the ultrafiltration and concentration liquid
2Solution is to final concentration 0.5mol/L, and alcohol concn 26% precipitates, centrifugal collection supernatant liquor; Supernatant liquor adopts the 100KD film to carry out ultrafiltration; Liquid adds calcium chloride solution to final concentration 0.5mol/L after the ultrafiltration, and ethanolic soln is to final concentration 80% precipitation polysaccharide, centrifugal collection polysaccharide precipitation; Polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively; The polysaccharide of washing obtains the refining Y group meningitis cocci polysaccharide of 3.56g through lyophilize.
Every calibrating index is as follows:
Protein content: 0.24%
Nucleic acid content: 0.26%
O-acetyl content: 0.62mmol/g
Sialic acid content: 608mg/g
K
DMolecule less than 0.5 reclaims: 95.3%
Telling test: produce precipitation line with Y group meningitis cocci specific corrosioning anteserum
Endotoxin content: 4EU/ microgram polysaccharide
The comparison of the present invention and prior art:
| Prior art (cold phenol extraction+ethanol precipitation+ultracentrifugation) | The leading indicator of bacterial polysaccharides | ||
| Endotoxin content (EU/ microgram) | Protein content | Nucleic acid content | |
| 50～600 | 0.6～5％ | 0.6～2％ | |
| Embodiment 1 | 4 | 0.31％ | 0.42％ |
| Embodiment 2 | 1 | 0.22％ | 0.31％ |
| Embodiment 3 | 2 | 0.24％ | 0.26％ |
| Embodiment 4 | 10 | 0.31％ | 0.35％ |
| Embodiment 5 | 8 | 0.42％ | 0.39％ |
| Embodiment 6 | 4 | 0.34％ | 0.28％ |
| Embodiment 7 | 8 | 0.21％ | 0.25％ |
| Embodiment 8 | 8 | 0.32％ | 0.36％ |
| Embodiment 9 | 4 | 0.24％ | 0.26％ |
As can be seen from the above table, bacterial polysaccharides endotoxin content, protein content, the nucleic acid content of the present invention's preparation all are better than prior art, especially endotoxin content illustrates that far below prior art the present invention is a kind of novel method that obtains the low bacterial polysaccharides effect highly significant of endotoxin content.
Claims (7)
1, a kind of macroporous resin is used for the method for bacterial polysaccharides endotoxin removal, the steps include:
(1) adopt bio-reactor that bacterium is carried out fermentation culture;
(2) after the sterilization of logarithmic growth later stage, centrifugal collection supernatant liquor;
(3) in supernatant liquor, add cetyl trimethylammonium bromide, centrifugal collecting precipitation;
(4) add 1mol/LCaCl in the precipitation
2Solution dissociates, and adds ethanol to final concentration 25%, centrifugal collection supernatant liquor;
(5) supernatant liquor adds ethanol to 50～90%; Centrifugal collection polysaccharide precipitation;
(6) polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively;
(7) polysaccharide after the washing obtains rough polysaccharide through lyophilize;
(8) raw sugar is dissolved in the 1/10 saturated neutral sodium acetate soln by 5～50mg/ml, with cold phenol extraction 1～10 time, and centrifugal collection supernatant liquor;
(9) supernatant liquor adopts the cocktail buffer ultrafiltration that contains 1～15% calcium ion chelator and 0.1～2.0% tensio-active agent;
(10) in ultrafiltration and concentration liquid, add pretreated macroporous resin, adsorbed 4～8 hours;
Under the condition of (11) 2000～5000rpm centrifugal 10～60 minutes, collect supernatant liquor;
(12) supernatant liquor adopts water for injection, and 3～300KD film carries out ultrafiltration;
(13) add CaCl in the ultrafiltration and concentration liquid
2Solution is to final concentration 0.1-2mol/L, and alcohol concn 20～30% precipitates, centrifugal collection supernatant liquor;
(14) supernatant liquor adopts 3～300KD film to carry out ultrafiltration;
(15) liquid adds CaCl after the ultrafiltration
2Or NaCl solution is to final concentration 0.1-2mol/L, and ethanolic soln is to final concentration 50-90% precipitation polysaccharide, centrifugal collection polysaccharide precipitation;
(16) polysaccharide precipitation respectively washs 2 times with dehydrated alcohol, acetone respectively;
(17) Xi Di polysaccharide obtains the low refining polysaccharide of endotoxin content through lyophilize.
2, a kind of macroporous resin according to claim 1 is used for the method for bacterial polysaccharides endotoxin removal, it is characterized in that: described bacterium be following any: b type hemophilus influenzae, A group meningitis cocci, C group meningitis cocci, W
135Group meningitis cocci, Y group meningitis cocci, salmonella typhi.
3, a kind of macroporous resin according to claim 1 is used for the method for bacterial polysaccharides endotoxin removal, it is characterized in that: described macroporous resin is for having porous polystyrene-divinylbenzene pedestal, can for following any: D3520, X-5, HP20.
4, a kind of macroporous resin according to claim 1 is used for the method for bacterial polysaccharides endotoxin removal, it is characterized in that: described raw sugar concentration of ordinary dissolution is 30～50mg/ml, cold phenol extraction 6～10 times.
5, a kind of macroporous resin according to claim 1 is used for the method for bacterial polysaccharides endotoxin removal, it is characterized in that: described calcium ion chelator be following any: sodium ethylene diamine tetracetate, trisodium citrate, concentration are 8～15%.
6, a kind of macroporous resin according to claim 1 is used for the method for bacterial polysaccharides endotoxin removal, it is characterized in that: described tensio-active agent be following any: Triton X-100, Tween-20, Sodium desoxycholate, concentration are 1.2～2.0%.
7, macroporous resin according to claim 1 is used for the method for bacterial polysaccharides endotoxin removal, it is characterized in that: described ultra-filtration membrane molecular weight cut-off is 100～300KD.
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| CN101982469A (en) * | 2010-10-22 | 2011-03-02 | 东瑞（南通）医药科技有限公司 | Method for removing bacterial endotoxins in cephalosporin antibiotics by utilizing macroporous adsorption resins |
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| CN104525151A (en) * | 2014-12-02 | 2015-04-22 | 佛山市博新生物科技有限公司 | Endotoxin adsorbent used in hemoperfusion, and preparation method thereof |
| CN104525151B (en) * | 2014-12-02 | 2016-10-05 | 佛山市博新生物科技有限公司 | Endotoxin absorbent for hemoperfusion and preparation method thereof |
| CN105061629A (en) * | 2015-08-31 | 2015-11-18 | 成都欧林生物科技股份有限公司 | Group-A meningococcus capsule crude polysaccharide purification technology |
| CN111295450A (en) * | 2017-07-05 | 2020-06-16 | 创赏有限公司 | Polysaccharide purification for vaccine production using lytic enzymes, tangential flow filtration and multimodal chromatography |
| CN109021136A (en) * | 2018-07-30 | 2018-12-18 | 中国医学科学院医学生物学研究所 | A kind of preparation process of Hib b |
| CN110845636A (en) * | 2019-12-02 | 2020-02-28 | 兰州生物制品研究所有限责任公司 | Method for removing endotoxin in bacterial polysaccharide |
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