CN104530250A - Method for purifying pneumococcal capsular polysaccharide - Google Patents
Method for purifying pneumococcal capsular polysaccharide Download PDFInfo
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- CN104530250A CN104530250A CN201410767761.XA CN201410767761A CN104530250A CN 104530250 A CN104530250 A CN 104530250A CN 201410767761 A CN201410767761 A CN 201410767761A CN 104530250 A CN104530250 A CN 104530250A
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Abstract
The invention relates to a method for purifying pneumococcal capsular polysaccharide and belongs to the field of biological products. The method for purifying pneumococcal capsular polysaccharide comprises the following steps: cracking bacteria of a streptococcus pneumoniae inactivated and fermented liquid, carrying out clarification and ultra-filtration to obtain ultra-filtration concentration liquid, precipitating by using low alcohols and carrying out ultra-filtration, degrading protein and nucleic acid into micromolecule fragments through enzymic catalytic reaction under the specific reaction condition by using protease and nuclease, further finely purifying after purifications processes of ultra-filtration and chromatography in subsequent processes to obtain purified pneumococcal capsular polysaccharide. The method is mild in reaction condition, simple and convenient to operate; the process is liable to control; the defects of the prior art can be overcome; poisonous and harmful chemical reagents such as phenols, chloroform and acetone are not used; the method is environmentally friendly and friendly to operator; the prepared capsular polysaccharide is high in recovery rate while the content of protein and nucleic acid is reduced.
Description
Technical field
The invention belongs to biological technical field, specifically, belong to vaccines arts; The present invention relates to a kind of method of purified capsular polysaccharide, relate to a kind of method not using phenol extraction, purifying Streptococcus pneumoniae capsular polysaccharide particularly.
Background technology
Streptococcus pneumoniae is a kind of Main Pathogenic Bacteria causing the upper respiratory tract infection disease such as pneumonia class disease and otitis media, sinusitis paranasal sinusitis, trachitis, can cause central nervous system, heart valve and bone joint infection time serious by blood circulation.Current is reduction by more than 2 years old children and the streptococcic effective means of high-order infection population pneumonia infection by injection multivalence pneumonia capsular polysaccharide vaccine.
In streptococcus pneumoniae fermenting process, thalline is except synthesis capsular polysaccharide, synthesize the compositions such as a large amount of protein, nucleic acid simultaneously, as the major impurity composition existed in purifying capsular polysaccharide process, World Health Organization's eighties in 20th century (WHO) and " European Pharmacopoeia ", when pneumonia polysaccharide vaccine and pneumonia polysaccharide conjugate vaccine quality standard are formulated, has all carried out strict regulation to the protein content in the streptococcus pneumoniae capsular polysaccharide of purifying, nucleic acid content.
Because different serotypes streptococcus pneumoniae capsular polysaccharide chemical property is different, the protein synthesized in fermenting process, the contaminant characteristics such as nucleic acid and content are also distinguished to some extent, therefore, often kind of serotype capsular polysaccharide all needs with organic solvent (lower alcohol, chloroform, acetone, phenol etc.) fractional precipitation, enzymic digestion, the methods such as ultrafiltration remove the pollution of protein and nucleic acid, but, do not have a kind of general method can purifying 23 kinds of serotype streptococcus pneumoniae capsular polysaccharides so far, aborning, all adopt the technique of carrying out purifying for different serotypes capsular polysaccharide respectively.
For existing streptococcus pneumoniae capsular polysaccharide purifying process, in the pneumonia product of current listing (as 23 valency pneumococcal polysaccharide vaccines), in pneumococcal capsular polysaccharide purge process, the a large amount of phenol of many uses, chloroform, acetone etc. are for the organic solvent of human body and bad environmental, the shortcoming of this method is in organic solvent extraction process, concuss can cause polysaccharide to degrade, rupture or lose initial space conformation, and the polysaccharide immunogenic be purified is lost.These organic reagents need repeatedly to use in purge process simultaneously, not only increase operation steps and time, and increase the risk of crossed contamination.
Therefore, how to simplify the purifying process of capsular polysaccharide, reduce simultaneously or avoid the use of such poisonous and harmful chemical reagent, adopt more easy, gentle, the eco-friendly method purified capsular polysaccharide of one, while raising polysaccharide recovery, reduce the content of the impurity such as protein nucleic acid, be optimization, improve Streptococcus pneumoniae vaccine production technique, improving the industry development trend of pneumococcal polysaccharide vaccine quality, is also technical problem urgently to be resolved hurrily at present.
Summary of the invention
The object of the invention is to the defect overcoming prior art, a kind of purification process of streptococcus pneumoniae capsular polysaccharide of reactive phenol extracting is provided, thus purifying process is farthest simplified, and remain the efficiency of purifying and the immunogenicity of purified product, in method of the present invention, do not add the organic solvent that phenol etc. is larger to harm, thus high-recovery obtain stable capsular polysaccharide antigen while, high degree reduces the pollution of purifying process to environment, represents the new trend of bio-pharmaceuticals industry development.
Method of the present invention is by utilizing proteolytic enzyme and nuclease to be small molecule segment by protein and nucleolysis, is removed proteolytic enzyme, nuclease by purification steps such as subsequent ultrafiltration, is achieved through impurity such as the small protein of enzymolysis and nucleic acid.
Specifically, the invention provides a kind of method of purifying Streptococcus pneumoniae capsular polysaccharide, namely proteolytic enzyme and nuclease is utilized to be small molecule segment by enzymic catalytic reaction by protein and nucleolysis under specific reaction conditions, consummate further by the purifying process such as ultrafiltration, chromatography in subsequent technique, obtain the capsular polysaccharide meeting the pharmacopeia such as WHO, European Union, Britain.Phenol extraction is used repeatedly to remove compared with the purification process such as protein, chloroform with tradition, this technological reaction mild condition, easy and simple to handle, process are easier to control, overcome the defect of prior art, do not use the poisonous and harmful chemical reagent such as phenols, chloroform, acetone, to environment, personnel close friend, while acquisition capsular polysaccharide high-recovery, reduce protein and nucleic acid content.
Thus, the invention provides a kind of reactive phenol extracting, utilize proteolytic enzyme and nuclease to be small molecule segment by enzymic catalytic reaction by protein and nucleolysis, then consummate further by purifying, obtain purified capsular polysaccharide.
Specifically, the method of the present invention's purified capsular polysaccharide from pneumonia streptococcus fermented liquid is initial feed liquid with deactivation fermented liquid, alcohol precipitation supernatant liquor is obtained through clarification, ultrafiltration and alcohol for sedimentation, proteolytic enzyme and nuclease is utilized to remove protein and nucleic acid impurity further by enzymic catalytic reaction after ultrafiltration, consummate further by purifying process afterwards, obtain purified capsular polysaccharide.
More specifically, the method for purified capsular polysaccharide from pneumonia streptococcus fermented liquid of the present invention comprises following 5 steps:
(1) 23 S. pneumoniae serotypes ferments respectively, deactivation and purifying.Concrete operations are, after any one S. pneumoniae serotypes in 23 serotypes ferments, streptococcus pneumoniae, by after its fermentation liquor cracking deactivation, cell debris removal, ultrafiltration and concentration, obtains ultrafiltration and concentration liquid;
(2) in ultrafiltration and concentration liquid, add sodium salt, add lower alcohol, precipitation, then through centrifugal or filtration, collect rudimentary alcohol precipitation supernatant liquor, to described supernatant liquor ultrafiltration, obtain alcohol precipitation supernatant ultrafiltrated; Preferably, described sodium salt is sodium-acetate;
(3) in described alcohol precipitation supernatant ultrafiltrated, add nuclease respectively and proteolytic enzyme carries out ferment treatment, after enzymic catalytic reaction terminates endonuclease reaction liquid;
(4) endonuclease reaction liquid described in ultrafiltration and concentration is to remove nuclease, proteolytic enzyme and to cut the small molecular weight impurity such as oligonucleotide, polypeptide, Nucleotide, amino acid of process generation through enzyme, obtains enzyme and cut ultrafiltrated after ultrafiltration;
(5) described enzyme cuts ultrafiltrated carries out protein, nucleic acid, proteolytic enzyme and nuclease further removal through affine ion exchange chromatography, then through ultrafiltration, after Sterile Filtration, obtains the capsular polysaccharide stoste of each serotype of purifying, cryopreservation.
Specifically, in step (1) wherein, cell debris is removed and is realized by centrifugation.More particularly, in step (1) wherein, by S. pneumoniae serotypes 1, serotype 2, serotype 3, serotype 4, serotype 5, serotype 6B, serotype 7F, Serotype8, serotype 9V, serotype 9N, serotype 10A, serotype 11A, serotype 12F, serotype 14, serotype 15B, serotype 17F, serotype 18C, serotype 19A, serotype 19F, serotype 20, serotype 22F, the fermentation liquor cracking deactivation of serotype 23F or serotype 33F, cell debris removes (centrifugal), after ultrafiltration dialysis, obtain ultrafiltration and concentration liquid.
Particularly, the lower alcohol that step (2) wherein adopts is preferably selected from one or more in ethanol, 1-propyl alcohol, 2-propyl alcohol, n-butyl alcohol, 2-butanols, 2-methyl isophthalic acid-propyl alcohol, 2-methyl-2-propanol or propylene glycol.Preferably, step (2) wherein adds lower alcohol to volume percent final concentration in ultrafiltration and concentration liquid is 10 ~ 40%.Preferably, step (2) wherein adds sodium salt to mass percent final concentration in ultrafiltration and concentration liquid is 1 ~ 8%.Preferably, the sedimentation time in step (2) wherein, more than 4 hours, is more preferably precipitates overnight.Preferably, the temperature of the settling step in step (2) wherein controls, at 2-8 DEG C, to be more preferably 3-6 DEG C, most preferably is 4 DEG C.
Preferably, step (2) is that in ultrafiltration and concentration liquid, first add sodium salt to mass percent final concentration be 1 ~ 8%, add the lower alcohol of one or more be selected from ethanol, 1-propyl alcohol, 2-propyl alcohol, n-butyl alcohol, 2-butanols, 2-methyl isophthalic acid-propyl alcohol, 2-methyl-2-propanol or propylene glycol again, be 10 ~ 40% to volume percent final concentration, in 2-8 DEG C of precipitates overnight, centrifugal or collecting by filtration lower alcohol supernatant liquor, and to described supernatant liquor ultrafiltration, obtain alcohol precipitation supernatant ultrafiltrated.
Preferably, the nuclease added in step (3) be selected from non-restriction endonuclease and other restricted or non-limiting rnases or deoxyribonuclease one or more.
Preferably, the proteolytic enzyme added in step (3) be wherein selected from Proteinase K, trypsinase, papoid and subtilisin one or more.
Preferably, in step (3) wherein, the pH value regulating alcohol precipitation supernatant ultrafiltrated is 6.5-9.5, adds magnesium chloride solution and makes Mg
2+concentration is 0-10mM, adds nuclease in alcohol precipitation supernatant ultrafiltrated, makes the final concentration of nuclease be 0 ~ 50U/ml, ferment treatment 2-8h at 20-40 DEG C.
Preferably, in step (3) wherein, after nuclease process terminates, regulate the pH value through the feed liquid of nuclease process to be 6.5-9.5, add calcium chloride solution to Ca
2+concentration is 2-10mM, adds proteolytic enzyme, and to feed liquid, the final concentration of proteolytic enzyme is 5 ~ 50mg/L, and temperature of reaction is 20 DEG C-40 DEG C, ferment treatment 2-10h.
In step (3), in described alcohol precipitation supernatant ultrafiltrated, add nuclease (be specially in non-restriction endonuclease and other restricted or non-limiting rnases or deoxyribonuclease one or more) and proteolytic enzyme (be specially in Proteinase K, trypsinase, papoid and subtilisin one or more) respectively.Wherein, the final concentration of alcohol precipitation supernatant ultrafiltrated amplifying nucleic acid enzyme is 0 ~ 50U/ml, and pH value is 6.5-9.5,0-10mM Mg
2+, ferment treatment 2-8h; In feed liquid, the final concentration of proteolytic enzyme is 5 ~ 50mg/L, and pH value is 6.5-9.5, and temperature of reaction is 20 DEG C-40 DEG C, at 2-10mM Ca
2+carry out ferment treatment 2-10h under condition, after enzymic catalytic reaction terminates, obtain endonuclease reaction liquid.
More preferably, the final concentration of alcohol precipitation supernatant ultrafiltrated amplifying nucleic acid enzyme is 10 ~ 40U/ml, and pH value is 7.5-8.5,0-10mM Mg
2+, ferment treatment 3-6h; In feed liquid, the final concentration of proteolytic enzyme is 15 ~ 40mg/L, and pH value is 7.5-8.5, and temperature of reaction is 25 DEG C-35 DEG C, at 4-8mM Ca
2+carry out ferment treatment 4-6h under condition, after enzymic catalytic reaction terminates, obtain endonuclease reaction liquid.
Preferably, in step (4), ultrafiltration also concentrates described endonuclease reaction liquid to remove nuclease, proteolytic enzyme and to cut the small molecular weight impurity such as oligonucleotide, polypeptide, Nucleotide, amino acid of process generation through enzyme, obtains enzyme and cut ultrafiltrated after ultrafiltration.
Preferably, in step (5), described enzyme cuts ultrafiltrated carries out protein, nucleic acid, proteolytic enzyme and nuclease further removal through affine ion exchange chromatography, then through ultrafiltration, Sterile Filtration, obtains the capsular polysaccharide stoste of each serotype of purifying.More preferably, in step (5), obtained polysaccharide stoste after film sterile filtration, in 2-8 DEG C of preservation.
More preferably, the invention provides a kind of reactive phenol extracting, utilize proteolytic enzyme and nuclease to remove the streptococcus pneumoniae capsular polysaccharide of protein and nucleic acid, specifically, the method comprises the following steps:
1., after the fermentation liquor cracking deactivation of S. pneumoniae serotypes 1, serotype 2, serotype 3, serotype 4, serotype 5, serotype 6B, serotype 7F, Serotype8, serotype 9V, serotype 9N, serotype 10A, serotype 11A, serotype 12F, serotype 14, serotype 15B, serotype 17F, serotype 18C, serotype 19A, serotype 19F, serotype 20, serotype 22F, serotype 23F and serotype 33F, centrifugal segregation cell debris, ultrafiltration dialysis, obtain ultrafiltration and concentration liquid.
2. in ultrafiltration and concentration liquid, adding sodium salt to final concentration is 1 ~ 8%, adding lower alcohol (being specially one or more in ethanol, 1-propyl alcohol, 2-propyl alcohol, n-butyl alcohol, 2-butanols, 2-methyl isophthalic acid-propyl alcohol, 2-methyl-2-propanol or propylene glycol) is 10 ~ 40% to final concentration, after in 2-8 DEG C of precipitates overnight, centrifugal or collecting by filtration lower alcohol supernatant liquor, and to described supernatant liquor ultrafiltration, obtain alcohol precipitation supernatant ultrafiltrated.
3. in described alcohol precipitation supernatant ultrafiltrated, add nuclease (be specially in non-restriction endonuclease and other restricted or non-limiting rnases or deoxyribonuclease one or more) and proteolytic enzyme (be specially in Proteinase K, trypsinase, papoid and subtilisin one or more) respectively.Wherein, nuclease final concentration is 0 ~ 50U/ml, and pH value is 6.5-9.5,0-10mM Mg
2+, ferment treatment 2-8h; Proteolytic enzyme final concentration is 5 ~ 50mg/L, and pH value is 6.5-9.5, and temperature of reaction is 20 DEG C ~ 40 DEG C, at 2-10mMCa
2+carry out ferment treatment 2-10h under condition, after enzymic catalytic reaction terminates, obtain endonuclease reaction liquid.
4. endonuclease reaction liquid described in ultrafiltration and concentration is to remove nuclease, proteolytic enzyme or to cut the small molecular weight impurity such as oligonucleotide, polypeptide, Nucleotide, amino acid processing and produce through enzyme, obtains enzyme and cut ultrafiltrated after ultrafiltration.
5. enzyme described in cuts ultrafiltrated carries out protein, nucleic acid, proteolytic enzyme and nuclease further removal through affine ion exchange chromatography, then through ultrafiltration, Sterile Filtration, obtains the capsular polysaccharide stoste of each serotype of purifying, cryopreservation.
Most preferred embodiment provided by the present invention is as follows: adopt bio-reactor to ferment to specific S. pneumoniae serotypes; Grow to the logarithmic phase later stage with cracking agent sterilization and cracking thalline.After fermentating liquid volume is concentrated 5-10 times by the ultrafiltration and concentration being the film bag of 100KD by centrifugal and molecular weight cut-off, adding sodium salt makes its mass concentration be 1-8%, adding subcooled lower alcohol in advance, to make it volume percent final concentration be 10-40%, 2-8 DEG C staticly settles more than 4 hours, centrifugal recovery supernatant liquor, with the lower alcohol in damping fluid ultrafiltration displacement alcohol precipitation supernatant liquor and salt ion, obtain alcohol precipitation supernatant ultrafiltrated.Regulate alcohol precipitation supernatant ultrafiltrated pH to 6.5-9.5, add MgCl
2make its final concentration to 0-10mM, add nuclease and make its final concentration to stirring reaction 2-8h at 0-50U/ml, 20-40 DEG C, nuclease catalyzed reaction terminates; Adding proteolytic enzyme made its final concentration use ultrafiltration after 5-50mg/L, 20-40 DEG C of stirring reaction 2-10 hour.Ultrafiltrated is splined on affine ion exchange column, obtains polysaccharide stoste 2-8 DEG C of preservation after ultrafiltration after collecting chromatographic solution, sterile filtration.
As previously mentioned, the method of the present invention's purified capsular polysaccharide from pneumonia streptococcus fermented liquid eliminates adding of the organic reagent such as phenols, chloroform, acetone, ether conventional in traditional purifying capsular polysaccharide technique, but extremely small with the proteolytic enzyme being successfully applied to biological products, this kind of consumption of nucleic acid, the biological enzyme that human body is substantially harmless is substituted; Meanwhile proteolytic enzyme, nuclease are very easily degraded in physical environment, decrease the use of a large amount of toxic organic chemical reagent simultaneously, thus greatly reduce on the impact of environment; Invention replaces multistep poisonous and harmful hazardous chemical operation steps in streptococcus pneumoniae capsular polysaccharide purge process, save time cost, production cost while simplifying purification step, protect operative's environment and health; Optimize, improve Pnu-Imune 23 production technique, improve the industry development trend of pneumococcal polysaccharide vaccine quality, the each serotype capsular polysaccharide prepared by this purification process is utilized to meet the contaminant protein content of pneumococcal capsular polysaccharide and the quality standard of contaminant nucleic acid in WHO, European Union's pharmacopeia, British Pharmacopoeia, protein and nucleic acid impurities content reduce greatly, reach less than 1.0% respectively; The polysaccharide antigen rate of recovery reaches 40-66%, higher than the 30-40% rate of recovery utilizing phenol repeatedly extraction process; To the final detection that gained streptococcus pneumoniae capsular polysaccharide purified polysaccharide carries out proteolytic enzyme, nuclease remains, remaining respectively below Monitoring lower-cut in purified polysaccharide, can be applicable to the purifying of bacterial eapsular polysaccharide and supplies the later stage for the preparation of vaccine.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
Bio-reactor is adopted to ferment to streptococcus pneumoniae 1 type; Grow to logarithmic phase later stage final concentration be 0.1% sodium deoxycholate sterilization and cracking thalline.By 9000g centrifugal 50 minutes, the ultrafiltration and concentration of feed liquid is carried out by after concentrated 8 times of fermentating liquid volume with the film bag that molecular weight cut-off is 100KD, adding sodium-acetate makes its mass percent final concentration be 7%, adding subcooled 1-propyl alcohol in advance, to make it volume percent final concentration be 20%, 4 DEG C staticly settle 8h, centrifugal recovery supernatant liquor, with the 1-propyl alcohol in damping fluid ultrafiltration displacement alcohol precipitation supernatant liquor and salt ion, obtains streptococcus pneumoniae 1 type alcohol precipitation supernatant ultrafiltrated.Regulate alcohol precipitation supernatant ultrafiltrated pH to 8.5, add MgCl
2make its final concentration to 2mM, add Benzonase nuclease and make its final concentration to 30U/ml, at 40 DEG C, stirring reaction 4h, Benzonase nucleic acid enzyme digestion reaction terminates; Adding trypsinase makes its final concentration to 20mg/L, regulates pH to 8.5, adds calcium chloride solution to Ca
2+concentration is 2mM, and 40 DEG C of stirring reactions carried out ultrafiltration after 8 hours.Ultrafiltrated is splined on hydroxyapatite column, obtains polysaccharide stoste 2 DEG C preservation after ultrafiltration after collecting chromatographic solution, sterile filtration.
Adopt the processing parameter of the present embodiment to carry out continuous three batches of streptococcus pneumoniae 1 type polysaccharide purifications, see enzyme treatment process process batch 1, batches 2 and batches 3 for concrete batch.
Phenol extraction technique: adopt bio-reactor to ferment to streptococcus pneumoniae 1 type; Grow to logarithmic phase later stage final concentration be 0.1% sodium deoxycholate sterilization and cracking thalline.By 9000g centrifugal 50 minutes, the ultrafiltration and concentration of feed liquid is carried out by after concentrated 8 times of fermentating liquid volume with the film bag that molecular weight cut-off is 100KD, adding sodium-acetate makes its mass percent final concentration be 7%, adding subcooled ethanol in advance, to make it volume percent final concentration be 20%, 4 DEG C staticly settle 8h, centrifugal recovery supernatant liquor; In above-mentioned supernatant liquor, add cold ethanol to final concentration is 60%, abundant mixing, 4 DEG C leave standstill 22 hours, centrifugal collecting precipitation, precipitation 0.3mol/L sodium acetate redissolves, with equal-volume phenol-centrifugal extracting of sodium acetate saturated solution, water for injection ultrafiltration is carried out to extract, obtained polysaccharide stoste 2 DEG C preservation after sterile filtration.
Enzyme treatment process three batches and phenol extraction technique gained obtain purified polysaccharide and detect data in table 1.Wherein: identification adopts immune double diffusion method (" Chinese Pharmacopoeia " three annex VIII C) or immune turbidimetry.Alditol acid system detection method see " European Union's pharmacopeia " 7 editions 01/2008:20522, colorimetry; Method for detecting protein content see " Chinese Pharmacopoeia " three annex VI B, Lowry method; Nucleic acid content detection method see " Chinese Pharmacopoeia " three appendix II A, ultraviolet visible spectrophotometry; Total nitrogen content detection method see " Chinese Pharmacopoeia " three annex VI A, Kjeldahl determination; Phosphorus content detection method see " Chinese Pharmacopoeia " three annex VII A, colorimetry; O-acetyl content method see " European Union's pharmacopeia " 5 editions 01/2005:20520, colorimetry; The large submethod of polysaccharide molecule adopts size exclusive chromatography (" Chinese Pharmacopoeia " three annex III D) to detect.
Table 1 serotype 1 streptococcus pneumoniae polysaccharides stoste component concentration
Embodiment 2
Bio-reactor is adopted to ferment to streptococcus pneumoniae 2 type; Grow to the logarithmic phase later stage with cracking agent sterilization and cracking thalline.Be that the ultrafiltration and concentration of the film bag of 100KD is by after concentrated 8 times of fermentating liquid volume by centrifugal 50 minutes of 9000g and molecular weight cut-off, adding sodium-acetate makes its mass percent final concentration be 5%, adding subcooled 2-propyl alcohol in advance, to make it volume percent final concentration be 25%, 4 DEG C staticly settle 22h, centrifugal recovery supernatant liquor, with the 2-propyl alcohol in damping fluid ultrafiltration displacement alcohol precipitation supernatant liquor and salt ion, obtain alcohol precipitation supernatant ultrafiltrated.Regulate pH to 8.0, add MgCl
2make its final concentration to 3mM, add Benzonase nuclease and make its final concentration to 25U/ml, at 37 DEG C, stirring reaction 6h, Benzonase nuclease catalyzed reaction terminates; Regulate pH to 8.5, add Proteinase K and make its final concentration to 20mg/L, add calcium chloride solution to Ca
2+concentration is 5mM, and 37 DEG C of stirring reactions carried out ultrafiltration after 4 hours, and ultrafiltrated is splined on hydroxyapatite and carries out chromatography, obtains polysaccharide stoste 8 DEG C preservation after ultrafiltration after collecting chromatographic solution, sterile filtration.
Adopt above-mentioned processing parameter to carry out continuous three batches of streptococcus pneumoniae 2 type polysaccharide purifications, see enzyme treatment process process batch 1, batches 2 and batches 3 for concrete batch.
Phenol extraction technique: adopt bio-reactor to ferment to streptococcus pneumoniae 2 type; Grow to logarithmic phase later stage final concentration be 0.1% sodium deoxycholate sterilization and cracking thalline.By 9000g centrifugal 50 minutes, the ultrafiltration and concentration of feed liquid is carried out by after concentrated 8 times of fermentating liquid volume with the film bag that molecular weight cut-off is 100KD, adding sodium-acetate makes its mass percent final concentration be 5%, adding subcooled ethanol in advance, to make it volume percent final concentration be 25%, 4 DEG C staticly settle 22h, centrifugal recovery supernatant liquor; In above-mentioned supernatant liquor, add cold ethanol to final concentration is 55%, abundant mixing, 4 DEG C leave standstill 20 hours, centrifugal collecting precipitation, precipitation 0.3mol/L sodium acetate redissolves, with equal-volume phenol-centrifugal extracting of sodium acetate saturated solution, water for injection ultrafiltration is carried out to extract, obtained polysaccharide stoste 2 DEG C preservation after sterile filtration.
Enzyme treatment process three batches and phenol extraction technique gained obtain purified polysaccharide and detect data in table 2.Wherein: identification adopts immune double diffusion method (" Chinese Pharmacopoeia " three annex VIII C) or immune turbidimetry.Alditol acid system detection method see " European Union's pharmacopeia " 7 editions 01/2008:20522, colorimetry; Method for detecting protein content see " Chinese Pharmacopoeia " three annex VI B, Lowry method; Nucleic acid content detection method see " Chinese Pharmacopoeia " three appendix II A, ultraviolet visible spectrophotometry; Total nitrogen content detection method see " Chinese Pharmacopoeia " three annex VI A, Kjeldahl determination; Phosphorus content detection method see " Chinese Pharmacopoeia " three annex VII A, colorimetry; The large submethod of polysaccharide molecule adopts size exclusive chromatography (" Chinese Pharmacopoeia " three annex III D) to detect.
Table 2 serotype 2 streptococcus pneumoniae 3 batches of polysaccharide stock solution quality detected results
Embodiment 3
Bio-reactor is adopted to ferment to streptococcus pneumoniae 5 type; Grow to the logarithmic phase later stage with cracking agent sterilization and cracking thalline.By centrifugal 50 minutes of 9000g, centrifugal and ultrafiltration and concentration was by after concentrated 8 times of fermentating liquid volume, adding sodium-acetate makes its mass percent final concentration be 4%, adding subcooled ethanol in advance, to make it volume percent final concentration be 12.5%, 4 DEG C staticly settle 20 hours, centrifugal or filtered and recycled supernatant liquor, with the ethanol in damping fluid ultrafiltration displacement alcohol precipitation supernatant liquor and salt ion, obtain alcohol precipitation supernatant ultrafiltrated.Regulate pH to 7.0, add MgCl
2make its final concentration to 3mM, add Benzonase nuclease and make its final concentration to 30U/ml, at 37 DEG C, stirring reaction 6h, Benzonase nuclease catalyzed reaction terminates; Regulate pH to 7.5, add calcium chloride solution to Ca
2+concentration is 2mM, adds Proteinase K and makes its final concentration to 20mg/L, 37 DEG C of stirring reaction ultrafiltration after 6 hours.Ultrafiltrated is splined on hydroxyapatite and carries out chromatography, obtains polysaccharide stoste 6 DEG C preservation after ultrafiltration after collecting chromatographic solution, sterile filtration.
Adopt above-mentioned processing parameter to carry out continuous three batches of streptococcus pneumoniae 5 type polysaccharide purifications, see enzyme treatment process process batch 1, batches 2 and batches 3 for concrete batch.
Phenol extraction technique: adopt bio-reactor to ferment to streptococcus pneumoniae 2 type; Grow to logarithmic phase later stage final concentration be 0.1% sodium deoxycholate sterilization and cracking thalline.By 9000g centrifugal 50 minutes, the ultrafiltration and concentration of feed liquid is carried out by after concentrated 8 times of fermentating liquid volume with the film bag that molecular weight cut-off is 100KD, adding sodium-acetate makes its mass percent final concentration be 4%, adding subcooled ethanol in advance, to make it volume percent final concentration be 12.5%, 4 DEG C staticly settle 20h, centrifugal recovery supernatant liquor; In above-mentioned supernatant liquor, add cold ethanol to final concentration is 65%, abundant mixing, 4 DEG C leave standstill 16 hours, centrifugal collecting precipitation, precipitation 0.3mol/L sodium acetate redissolves, with equal-volume phenol-centrifugal extracting of sodium acetate saturated solution, water for injection ultrafiltration is carried out to extract, obtained polysaccharide stoste 2 DEG C preservation after sterile filtration.
Enzyme treatment process three batches and phenol extraction technique gained obtain purified polysaccharide and detect data in table 3.Detection method is with embodiment 1,2.
Table 3 serotype 5 streptococcus pneumoniae polysaccharides stoste component concentration
Embodiment 4
Bio-reactor is adopted to ferment to streptococcus pneumoniae 6B type; Grow to the logarithmic phase later stage with cracking agent sterilization and cracking thalline.By centrifugal 50 minutes of 9000g, centrifugal and molecular weight cut-off was that the ultrafiltration and concentration of the film bag of 100KD is by after concentrated 8 times of fermentating liquid volume, adding sodium-acetate makes its mass percent final concentration be 6%, adding subcooled 1-propyl alcohol in advance, to make it volume percent final concentration be 30%, 4 DEG C staticly settle 22 hours, centrifugal recovery supernatant liquor, with the 1-propyl alcohol in damping fluid ultrafiltration displacement alcohol precipitation supernatant liquor and salt ion, obtain alcohol precipitation supernatant ultrafiltrated.Regulate pH to 7.2, add MgCl
2make its final concentration to 3mM, add Benzonase nuclease and make its final concentration to 20U/ml, at 37 DEG C, stirring reaction 6h, Benzonase nuclease catalyzed reaction terminates; Regulate pH to 7.0, add calcium chloride solution to Ca
2+concentration is 2mM, and add Proteinase K and make its final concentration to 20mg/L, 37 DEG C of stirring reactions used ultrafiltration after 6 hours.Ultrafiltrated is splined on hydroxyapatite chromatography post, obtains polysaccharide stoste 4 DEG C preservation after ultrafiltration after collecting chromatographic solution, sterile filtration.
Adopt above-mentioned processing parameter to carry out continuous three batches of streptococcus pneumoniae 6B type polysaccharide purifications, see enzyme treatment process process batch 1, enzyme treatment process process batches 2 and enzyme treatment process process batches 3 for concrete batch.
Phenol extraction technique: adopt bio-reactor to ferment to streptococcus pneumoniae 2 type; Grow to logarithmic phase later stage final concentration be 0.1% sodium deoxycholate sterilization and cracking thalline.By 9000g centrifugal 50 minutes, the ultrafiltration and concentration of feed liquid is carried out by after concentrated 8 times of fermentating liquid volume with the film bag that molecular weight cut-off is 100KD, adding sodium-acetate makes its mass percent final concentration be 6%, adding subcooled ethanol in advance, to make it volume percent final concentration be 30%, 4 DEG C staticly settle 22h, centrifugal recovery supernatant liquor; In above-mentioned supernatant liquor, add cold ethanol to final concentration is 50%, abundant mixing, 4 DEG C leave standstill 20 hours, centrifugal collecting precipitation, precipitation 0.3mol/L sodium acetate redissolves, with equal-volume phenol-centrifugal extracting of sodium acetate saturated solution, water for injection ultrafiltration is carried out to extract, obtained polysaccharide stoste 2 DEG C preservation after sterile filtration.
Enzyme treatment process three batches and phenol extraction technique gained obtain purified polysaccharide and detect data in table 3.
Table 4 serotype 6B streptococcus pneumoniae 3 batches of polysaccharide stock solution quality detected results
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. a method for purifying Streptococcus pneumoniae capsular polysaccharide, is characterized in that, utilizes proteolytic enzyme and nuclease to be small molecule segment by enzymic catalytic reaction by protein and nucleolysis, then consummate further by purifying, obtains purified capsular polysaccharide.
2. the method for claim 1, it is characterized in that, be initial feed liquid with streptococcus pneumoniae deactivation fermented liquid, alcohol precipitation supernatant liquor is obtained through clarification, ultrafiltration and alcohol for sedimentation, proteolytic enzyme and nuclease is utilized to be small molecules by degrade further macro-molecular protein and nucleic acid impurity of enzymic catalytic reaction after ultrafiltration, ultrafiltration is removed, rear consummate further by purifying, obtains purified capsular polysaccharide.
3. method as claimed in claim 2, is characterized in that, comprise the following steps:
(1) use cracking agent sterilization cracking thalline, by the fermented liquid cracking deactivation of streptococcus pneumoniae, remove cell debris, then after ultrafiltration and concentration, obtain ultrafiltration and concentration liquid;
(2) in ultrafiltration and concentration liquid, first add inorganic salt, add lower alcohol, precipitation, then through the centrifugal or rudimentary alcohol precipitation supernatant liquor of collecting by filtration, to the ultrafiltration of lower alcohol supernatant liquor, obtain alcohol precipitation supernatant ultrafiltrated; Described inorganic salt are sodium salt or calcium salt;
(3) in alcohol precipitation supernatant ultrafiltrated, add nuclease and proteolytic enzyme carries out ferment treatment, after enzymic catalytic reaction terminates endonuclease reaction liquid;
(4) endonuclease reaction liquid described in ultrafiltration and concentration is to remove nuclease, proteolytic enzyme and to cut other small molecular weight impurity processing and produce through enzyme, obtains enzyme and cut ultrafiltrated after ultrafiltration;
(5) enzyme cuts ultrafiltrated carries out the protein, nucleic acid, proteolytic enzyme and the nuclease that remain removal through affine ion exchange chromatography, then through ultrafiltration, after Sterile Filtration, obtains the streptococcus pneumoniae capsular polysaccharide stoste of purifying, cryopreservation.
4. method as claimed in claim 3, it is characterized in that, the pneumonia streptococcus fermented liquid of step (1) is wherein the fermented liquid of S. pneumoniae serotypes 1, serotype 2, serotype 3, serotype 4, serotype 5, serotype 6B, serotype 7F, Serotype8, serotype 9V, serotype 9N, serotype 10A, serotype 11A, serotype 12F, serotype 14, serotype 15B, serotype 17F, serotype 18C, serotype 19A, serotype 19F, serotype 20, serotype 22F, serotype 23F or serotype 33F.
5. method as claimed in claim 3, it is characterized in that, the lower alcohol that step (2) wherein adopts is selected from one or more in ethanol, 1-propyl alcohol, 2-propyl alcohol, n-butyl alcohol, 2-butanols, 2-methyl isophthalic acid-propyl alcohol, 2-methyl-2-propanol or propylene glycol.
6. method as claimed in claim 3, it is characterized in that, it is 10% ~ 40% that step (2) adds lower alcohol to volume percent final concentration in ultrafiltration and concentration liquid.
7. method as claimed in claim 3, it is characterized in that, step (2) adds sodium salt in ultrafiltration and concentration liquid or calcium salt to mass percent final concentration is 1% ~ 8%.
8. method as claimed in claim 3, is characterized in that, after step (2) adds lower alcohol, in 2-8 DEG C of precipitation more than 4 hours.
9. method as claimed in claim 3, is characterized in that, the nuclease added in step (3) be wherein selected from non-restriction endonuclease and other restricted or non-limiting rnases, deoxyribonuclease one or more.
10. method as claimed in claim 3, is characterized in that, the proteolytic enzyme added in step (3) be wherein selected from Proteinase K, trypsinase, papoid and subtilisin one or more.
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