CN102660602A - Method for rapidly purifying bacteria capsular polysaccharide - Google Patents
Method for rapidly purifying bacteria capsular polysaccharide Download PDFInfo
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Abstract
The invention discloses a method for rapidly purifying bacteria capsular polysaccharide, which can rapidly remove the pollutants in a specific bacteria broth, the capsular polysaccharide comprises protein and nucleic acid, the purified capsular polysaccharide can be reserved, fermentation of capsular polysaccharide-containing bacteria is included, acid is added for adjusting the pH value of the deposing thalline and impurity in a broth, the solution is subjected to microfiltration, ultrafiltration concentration and filter washing to obtain a crude bacteria capsular polysaccharide solution. A basic principle of the present invention is characterized in that acid is added to regulate the pH value to a range of 3-5, thalline with complete form and other impurities can be removed, and the capsular polysaccharide is purified in the solution. The capsular polysaccharide can be used for purifying pneumococcus, b-type haemophilus influenza, epidemic meningococcus and typhoid bacillus.
Description
Technical field:
The present invention relates to field of biological pharmacy, relate in particular to and a kind ofly can from ferment product, remove pollutent, the method for purifying capsular polysaccharide.
Background technology:
Streptococcus pneumoniae, b type hemophilus influenzae, epidemic meningitis coccus and Corynebacterium diphtheriae are the important pathogenic bacterias that threatens the human life.It is the target that medical circle is struggled for many years that the exploitation vaccine prevents the infection of these germs.From the sixties in last century, find that through laboratory facilities these bacteriums have common morphological structure characteristics, be exactly that surface of cell membrane is wrapped in one deck pod membrane, this layer pod membrane is to be wrapped in each surface of cell membrane by polysaccharide to form, so claim capsular polysaccharide.In human body, pod membrane is for the pathogenic decisive role that plays of these bacteriums, because, being adsorbed onto on the bacterial film through preventing antibody, pod membrane can disturb the phagolysis of immunocyte to bacterium, makes bacterium to breed in vivo and causes a disease.Discover that the bacterial capsule polysaccharide has good immunogenicity as vaccine, can stimulate adult to produce antibody and resist infection with capsular polysaccharide bacterium with sound immunologic function; But, because children's below 2 years old immunity system is grown imperfection, capsular polysaccharide is not had immunne response, and this crowd receiving these pathogenic bacterium to threaten the most serious crowd, it is the emphasis problem of medical circle that exploitation can be protected this crowd's vaccine.From the eighties in last century; Discover, when capsular polysaccharide with covalent linkage and protein bound, behind Toxoid,tetanus, DT or CRM197 (variation diphtheria anatoxine); Form the polysaccharide-protein combined vaccine; Can produce the immune response that is directed to capsular polysaccharide in the following infants at 2 years old, since then, a kind of synthetic vaccine birth that is attached to protein carrier with the bacterial capsule polysaccharide.
At present, on market, be used to prevent the 23 valency pneumococcal polysaccharide vaccines that have of grownup streptococcus pneumoniae coccus infection, and 7 valencys of prevention in children, 10 valencys and 13 valency pneumococcal Polysaccharide Conjugate Vaccines are widely used in the world.In addition, typhoid fever (vi) also use for many years by polysaccharide vaccine, b type hemophilus influenzae polysaccharide conjugate vaccine, 4 valency epidemic meningitis polysaccharide conjugate vaccines.
In view of chemical structure; Different bacteriums has different capsular polysaccharides; And different capsular polysaccharides is also arranged with a kind of bacterium different serotypes bacterium; Like streptococcus pneumoniae 90 kinds of different serotypes are arranged, meningococcus has A, B, C, W135 and Y crowd, and they all have the capsular polysaccharide of chemical structures.Discover that every kind of capsular polysaccharide has strict repeated chemical structure, this chemical structure can or contain the oligosaccharide that 7-8 monosaccharide residue the most nearly forms by a monosaccharide units to be formed.This repeated unit can be linearity or contains the bifurcation structure of non-carbohydrate substituted radical, and these non-carbohydrate substituted radicals can be O-ethanoyl (O-acetyl), Phosphoric acid glycerol esters (glycerol phosphate) or pyruvic acid (pyruvate); In addition, some capsular polysaccharides of streptococcus pneumoniae possibly contain uncommon monosaccharide residue, like diamino-(diamino-), and deoxidation and branch fork chain sugar etc.This bacterial polysaccharides diversity structure has brought challenge and difficulty for these polysaccharide methods of purifying.
Prepare vaccine with capsular polysaccharide, comprise polysaccharide vaccine or polysaccharide conjugate vaccine, all need prepare as raw material with the capsular polysaccharide that meets the certain mass standard.In order to obtain highly purified capsular polysaccharide; The liquid medium of available being rich in nutrition composition comes fermenting bacterial; And then from fermented liquid the purifying capsular polysaccharide, the principal pollutant that will remove in the purge process comprise bacterioprotein, nucleic acid and medium component; If preparation also need be controlled the pollution of C-polysaccharide during pneumococcal capsular polysaccharide.
The years of development process has been experienced in the foundation of bacterial capsule polysaccharide purification technology; In the traditional method purifying capsular polysaccharide process; The pollution of nucleic acid of removing in the polysaccharide soln is relatively more difficult to reach the required purity requirement of vaccine preparation; Because the chemical property of the capsular polysaccharide of different bacterium is different, every kind of bacterial capsule polysaccharide need remove pollution of nucleic acid with special methods, does not have the capsular polysaccharide that a kind of method can other bacterium of purifying.Some bacterial capsule polysaccharide need precipitate through a large amount of ethanol, simultaneously, use different organic solvent extractions, like ether etc., helps purifying.Ethanol sedimentation can be removed the protein pollutant in the polysaccharide usually effectively, and its concentration is reduced to lower level; But; Be difficult to be reduced to the level that the vaccine that is used for injection requires, comprise and adopt organic solvent chloroform and butanols mixed solvent or phenol and polysaccharide soln mixing; Jolt 4-6 hour; Then, use low-speed centrifugal, accumulate between water and the organic phase metaprotein can with the separation of polysaccharides of aqueous phase.But the shortcoming of this method is in extraction process, and polysaccharide can be degraded, and fracture perhaps loses its luv space conformation, and the polysaccharide that purifying comes out has not been effective immunogen.Because the polytropy of different bacterium polysaccharide structures has limited the validity of these separation methods.Thus, reaching a conclusion is not have a kind of method capsular polysaccharide of purifying different bacterium effectively in the early stage traditional method.
However; There are several kinds to be purified into high purity and to preserve its immunogenic different bacterium capsular polysaccharide method and invented; Particularly when purifying different serotypes pneumococcal capsular polysaccharide, these methods can be purified into effectively and comprise 24 kinds of serotype capsular polysaccharides.Comprise 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F, and be used to prepare the capsular polysaccharide raw material of 23 valency pneumococcal polysaccharide vaccines.
1980; The patent US4242501 of U.S. American Cyanamid Company company has set forth a kind of purifying pneumococcal capsular polysaccharide method; On this patent basis; The said firm has registered another patent US4686102 again in 1987 subsequently, in order to 23 different serotype pneumococcal capsular polysaccharides of purifying.After the basic technology approach of this patent is the streptococcus pneumoniae fermentation; Add Sodium desoxycholate (sodium deoxycholate) dissolution of bacteria; Capsular polysaccharide on the thalline is released in the fermented liquid; Centrifugal removed cell debris after, with ethanol sedimentation twice, this step can be removed a large amount of pollutents that comprise protein; Polysaccharide soln behind adding cetyl trimethylammonium bromide to the ethanol sedimentation is further removed pollutent.Through control cetyl trimethylammonium bromide concentration, can get off by the most of serotype polysaccharide precipitation of adsorption precipitation, these deposition polysaccharide can be dissolved in sodium chloride solution, those undissolved macromolecule contaminants of centrifugal removal subsequently.But, have four kinds of serotype polysaccharide to form deposition with cetyl trimethylammonium bromide, the effect that adds cetyl trimethylammonium bromide is the deposit fouling thing, these pollutents of centrifugal removal.Subsequently, add ethanol again to remove cetyl trimethylammonium bromide.Centrifugal removal post precipitation adds the residual pollutent of charcoal absorption, like albumen and nucleic acid.At last, obtain the purifying capsular polysaccharide through dialysis.Visible from technological process, although this method has been set up the purifying platform of a pneumococcal capsular polysaccharide, to use with a kind of method and can be purified into the different serotypes polysaccharide, step is numerous and diverse, should not grasp; Wherein also comprised multistep ethanol sedimentation, centrifugal, time-consuming, cost of equipment is high; And should not enlarge production.Cetyl trimethylammonium bromide settling step technical sophistication, the different serotypes treatment process is different.In addition; The polysaccharide that final purifying comes out only can satisfy the quality standard of preparation 23 valency pneumonia polysaccharide vaccines; Be that albumen and nucleic acid content are lower than 2%-7%, can't satisfy existing pneumococcal Polysaccharide Conjugate Vaccine albumen and nucleic acid content in the polysaccharide raw material are lower than 1% specification of quality.
To 1998, the patent US5714354 of U.S. American Home Products company proposed a kind ofly not have ethanol sedimentation and come improving one's methods of purifying pneumococcal capsular polysaccharide.This method is through precipitate 20 capsular polysaccharides in 23 kinds of streptococcus pneumoniae serotypes with cetyl trimethylammonium bromide; Use activated carbon filtration, hydroxyl phosphoric acid grey matter salt (Hydroxyapatite) chromatography and anion-exchange chromatography to come the pollutent in the further separating polyose then, this purifying process step can be purified into most of serotype capsular polysaccharide.And other 3 can not with the purifying of the sedimentary serotype of cetyl trimethylammonium bromide (serotype 7F, 14 and 33F), be to adopt modifying method to carry out purifying, comprise the anionite-exchange resin chromatography.Compare with last patent purifying process, this purifying process can save for 75% purifying time, and expense also reduces, and does not need specific installation.The quality of all 23 serotype capsular polysaccharides that this method purifying comes out reaches the quality index of polysaccharide vaccine.
Coexisted 1998, the patent US5847112 of U.S. Merck company also issues a kind of method of purifying pneumonia capsular polysaccharide.Basic skills is for to come separating polyose from the bacterium liquid that ferments brokenly in two steps with ethanol; The first step is to come sedimentation cell fragment and pollutent with the ethanol that hangs down per-cent, like the C-polysaccharide, keeps polysaccharide in solution; Next, the ethanol that adds high density precipitates capsular polysaccharide, and other pollutent of staying in the solution is abandoned.After will precipitating polysaccharide dissolving, use ordinary method again, such as nucleic acid and protease digestion, perhaps organic solvent further removes the albumen and the nucleic acid of pollution, and rough polysaccharide is by ethanol sedimentation and freeze-drying acquisition.Subsequently, obtain full polysaccharide of purified or partially-hydrolyzed polysaccharide through the anion-exchange chromatography purifying.Although the polysaccharide that comes out with above two kinds of method purifying can reach the higher purity standard,, step is still numerous and diverse, and processing condition control requires high.The rapid precipitate and separate step length consuming time of multistep particularly.
The patent US20060228381 of U.S. Wyeth company has proposed a kind of method of more succinct purifying pneumococcal capsular polysaccharide.Basic Ways is with behind the beans substratum fermentation streptococcus pneumoniae; Add Sodium desoxycholate and dissolve thalline; Add acid-conditioning solution pH to 5.5 then with bulk deoxidation Sodium cholic acid and the most soluble property albumen of getting off, remove cell debris and throw out in the solution through centrifuging and ultrafiltration.Thus obtained rough polysaccharide still needs traditional method to be further purified, and comprise with activated carbon filtration, hydroxyapatite column absorption remaining impurities, and the ultrafiltration filter wash obtains refining polysaccharide.Like this characteristics of purification process be through broken this step of fermented liquid of acidifying with most pollutent, like albumen, nucleic acid, intracellular toxin etc., separate with capsular polysaccharide in the solution, simplified subsequent technique, shortened the purifying time.But; Because what be to use carries out purifying with the Sodium desoxycholate lysate; The C-glycocalix that contains in the cell walls of some serotype discharges; Because the chemical property of C-polysaccharide and capsular polysaccharide is similar, is difficult in purifying process process subsequently, being removed, caused in the purified polysaccharide C-polysaccharide content too high.
The present invention has proposed a kind of purification process more fast through having summed up the purification process of existing bacterial capsule polysaccharide, and each item standard of the purifying capsular polysaccharide that is obtained has all reached existing WHO quality standard.
Summary of the invention:
The method that the purpose of this invention is to provide a kind of fast purifying bacterial capsule polysaccharide, it can remove pollutent wherein fast from specify ferment product, keep the capsular polysaccharide solution of purifying.
The present invention includes following steps:
Step 1, the bacterium that goes out to produce capsular polysaccharide in the fermentation cylinder for fermentation that bacteria culture medium is housed;
Thalline in the step 2, centrifugal and/or micro-filtration fermented liquid is collected the solution that no insoluble particle contains capsular polysaccharide;
Step 3 concentrates the micro-filtrate that contains capsular polysaccharide with filter wash with ultra-filtration membrane;
Step 4 adds acid to liquid concentrator, regulates and contains capsular polysaccharide pH value of solution value≤5 scopes and come precipitated impurities;
Step 5, centrifugal and/or micro-filtration is removed the throw out in the solution;
Step 6, ultrafiltration filter wash polysaccharide soln obtains the extremely low capsular polysaccharide solution of impure content.
In the method for above-mentioned fast purifying bacterial capsule polysaccharide, said bacterium comprises: streptococcus pneumoniae, b type hemophilus influenzae, epidemic meningitis coccus and Corynebacterium diphtheriae.
In the method for above-mentioned fast purifying bacterial capsule polysaccharide, added acid is any one in phosphoric acid, acetic acid, hydrochloric acid, sulfuric acid or the nitric acid in step 4.
In the method for above-mentioned fast purifying bacterial capsule polysaccharide, the scope of pH value is 3.0～5.0 in step 4.
In the method for above-mentioned fast purifying bacterial capsule polysaccharide, the membrane pore size of the used microfiltration membrane of micro-filtration is any one among 0.22,0.45,0.65, the 1.0 μ m.
In the method for above-mentioned fast purifying bacterial capsule polysaccharide, the used ultra-filtration membrane of ultrafiltration is that molecular weight is any one in 5kD to the 500kD scope.
In the method for above-mentioned fast purifying bacterial capsule polysaccharide, the used solution of filter wash is pure water, damping fluid or salts solution or their mixed solution.
In the capsular polysaccharide solution that is obtained through above-mentioned purifying process process, comprise that soluble proteins and nucleic acid foreign matter content all show the reduction that lands.
The present invention can be used for purified polysaccharide from contain the capsular polysaccharide bacterium, just for example, and is not limited to these bacteriums here, like the streptococcus pneumoniae different serotypes, comprises 1,2,3,4; 5,6A, 6B, 7F, 8,9N, 9V, 10A; 11A, 12F, 14,15B, 17F, 18C, 19A; 19F, 20,22F, 23F and 33F, b type hemophilus influenzae, epidemic meningitis coccus, and Corynebacterium diphtheriae.
In the present invention, it is core that step 4 adds Acid precipitation impurity, promptly through add phosphoric acid with the liquid concentrator pH regulator between the 3.0-5.0, come protein precipitation and nucleic acid with this scheme.
In the present invention, adopt spinning, micro-filtration and ultrafiltration and concentration three-step approach to remove throw out and insoluble particle better effects if in the step 2 fermented liquid, different according to bacterial species; Perhaps pneumococcal serotype is different; The centrifuged supernatant of selecting for use 0.22,0.45,0.65 or 1.0 μ m films to come micro-filtration to obtain; Remove residual cell granulations and fragment in the solution, and insoluble particles; Also can omit centrifugal this step, and directly fermented liquid carried out micro-filtration, to remove thalline and the cell debris in the fermented liquid; With 30 to 500kD films micro-filtrate is carried out ultrafiltration and concentration; And come the ultrafiltration cleaning solution with deionized water or damping fluid; Remove the small molecules pollutent; Through the purification process of these steps, can remove 90-98% albumen and 95-99% nucleic acid in the capsular polysaccharide solution, make the throw out in the fermented liquid remove more thorough.
Embodiment:
A kind of method of fast purifying bacterial capsule polysaccharide; With traditional from broken bacterium/or bacteriolyze after ferment product carry out purification process main difference be; The present invention is through the pollutent in the solution being removed fast and easily, being kept the capsular polysaccharide of the complete molecular weight in the solution.The present invention can be used for, like streptococcus pneumoniae, and b type hemophilus influenzae, the purifying of epidemic meningitis coccus and Corynebacterium diphtheriae capsular polysaccharide, foreign protein and nucleic acid content can reduce 90-98% in the polysaccharide soln behind the purifying.The purifying capsular polysaccharide technology that adopts at present is numerous and diverse, contains the precipitate and separate step of multistep, is difficult for large-scale industrialization production, needs the input and the manpower of valuable in a large number equipment to operate.
The purifying process of bacterial capsule polysaccharide is through semicentennial development transition, and along with novel biomaterial is constantly released, the macromolecule purifying technology is also improved thereupon, to adapt to the standard that the biotechnological formulation quality is improved constantly.For the vaccine for preparing with the bacterial capsule polysaccharide; Like pneumococcal polysaccharide vaccine and multivalent pneumococcal polysaccharide combined vaccine; B type hemophilus influenzae combined vaccine, meningococcal polysaccharide vaccine and polysaccharide conjugate vaccine, and Typhoid Vi Polysaccharide Vaccine and Vi polysaccharide conjugate vaccine; The quality standard of its capsular polysaccharide is also in continuous improve; Impurity protein content permissible criterion with in the polysaccharide is an example, and one of the quality standard of 23 valency pneumococcal polysaccharide vaccines is that requirement impurity albumen and nucleic acid standard are lower than 2-7% from the eighties in last century, and the capsular polysaccharide raw materials quality standard used to various different bacterium polysaccharide conjugate vaccines in 2000 is to be lower than 1%.The purifying process of bacterial capsule polysaccharide is also from early stage nearly 16 road technologies; Be improved to the 10 road main technique steps that adopting at present; Shorten time and cost that purifying needs, simultaneously, also reached modern polysaccharide vaccine preparation required standard qualitatively.
The present invention is on the basis that the intermediate product to existing bacterial capsule polysaccharide purification process procedure detects and assesses, and further optimizes and the purifying process platform set up, can be purified into the capsular polysaccharide of different bacterium fast, easily, and step is following:
The present invention includes following steps:
Step 1, the bacterium that goes out to produce capsular polysaccharide in the fermentation cylinder for fermentation that bacteria culture medium is housed;
Thalline in the step 2, centrifugal and/or micro-filtration fermented liquid is collected the solution that no insoluble particle contains capsular polysaccharide;
Step 3 concentrates the micro-filtrate that contains capsular polysaccharide with filter wash with ultra-filtration membrane;
Step 4 adds acid to liquid concentrator, regulates and contains capsular polysaccharide pH value of solution value≤5 scopes and come precipitated impurities;
Step 5, centrifugal and/or micro-filtration is removed the throw out in the solution;
Step 6, ultrafiltration filter wash polysaccharide soln obtains the extremely low capsular polysaccharide solution of impure content.
Through above six purifying process steps; Can be with the major impurity in the solution, like albumen and nucleic acid, content reduce 90-98%; Simultaneously, the immunogenicity of the capsular polysaccharide that comes out of purifying, the integrity of molecule all are superior to or are equal to the polysaccharide that current purification method obtains.
Step 7 adopts prior art that the bacterial capsule polysaccharide is carried out subsequent purification.
The principle of design of the method for the purification of bacterial capsular polysaccharide that the present invention proposes and the control of test conditions are following:
(1), the control of fermentation using bacteria process
Be purified into highly purified capsular polysaccharide, need control, particularly on fermenting step, should reduce pollutent as far as possible and enter into the fermented liquid that is used for purified polysaccharide each link that possibly pollute in the polysaccharide production process; Wherein, The foreign-matter contamination that discharges owing to bacterial cell disruption, like albumen and nucleic acid, and the impurity that contains in the substratum; Comprise albumen, nucleic acid and impurity polysaccharide etc. is the main source of pollutent.With the streptococcus pneumoniae is example, and this bacterium is a gram-positive microorganism, can tolerate oxygen, and its form has multifarious characteristics, can between smooth (Transparent) and coarse (Opaque) colonial morphology, transform.When being in smooth bacterium attitude, bacterium surface only has a spot of capsular polysaccharide, is the form that human body nasal cavity endophyte falls to existing; And the bacterium surface that is in the rough bacteria attitude contains a large amount of capsular polysaccharides, is the form that the streptococcus pneumoniae in the blood of human body exists, and the existence of capsular polysaccharide can stop the host immune cytophagy, makes bacterium to survive and breeding.
Experiment is illustrated in the liquid nutrient medium, and the part capsular polysaccharide of streptococcus pneumoniae surface parcel can be shed in the fermented liquid, can be used for the purifying capsular polysaccharide.Obviously, obtain capsular polysaccharide from the thalline surface, must be with obtaining behind the bacterial cell disruption, this just makes pollutents such as impurity albumen in the bacterial body, nucleic acid all be released in the fermented liquid, thereby has increased the difficulty of purified polysaccharide.For fear of this problem; Simultaneously, according to pneumococcal cultural characters, the present invention at first adopts strict anaerobic fermentation; Make that streptococcus pneumoniae is main with the rough bacteria attitude in fermented liquid; With this understanding, streptococcus pneumoniae can produce a large amount of capsular polysaccharides, and the part capsular polysaccharide can be released into the nutrient solution from bacterium surface.In the pH value (7.0-7.6) in neutral range of control fermented liquid, polysaccharide can stably exist in solution and not degrade.On the other hand, because streptococcus pneumoniae contains N,O-Diacetylmuramidase, after bacterial death, can discharge this enzyme self-dissolving; Therefore, to stop fermentation in time, gather in the crops at fermentation using bacteria to growth platform after date; So that the minimizing bacterial death discharges entocyte, the situation of polluting fermented liquid takes place.
The pneumovax that uses in the market; Comprise 23 valency pneumonia polysaccharide vaccines and 13 valency pneumococcal Polysaccharide Conjugate Vaccines; And b type hemophilus influenzae polysaccharide conjugate vaccine; Producing used capsular polysaccharide raw material all is to dissolve thalline by adding in tensio-active agent to the fermented liquid; Capsular polysaccharide to discharge on the bacteria cell wall carries out purifying, and in fact the source of final capsular polysaccharide has two approach, the polysaccharide that promptly after bacterium surface drops to polysaccharide and the bacteriolyze the fermented liquid, is discharged by cell walls during the fermentation.Although when taking the purifying capsular polysaccharide in this way, initial polysaccharide total amount will be more than the initial amount of purifying capsular polysaccharide from the fermented liquid supernatant liquid of removing thalline only; But; Impurity component content in the bacteriolyze fermented liquid in the starting soln will be far above fermented liquid supernatant liquid, and this has increased burden for the subsequent purification operation, needs more step to carry out purifying; Therefore; On the one hand the polysaccharide in the solution can increase in loss in too much purification step, and its yield and from fermented supernatant fluid, carry out purifying and compare is not preponderated; On the other hand, add after tensio-active agent comes bacteriolyze, although can discharge more polysaccharide,, be present in the impurity polysaccharide component of cell walls, also be released out such as the C-polysaccharide.And the chemical property of C-polysaccharide is similar with capsular polysaccharide, in follow-up purge process, removes very difficulty.The pollution of C-polysaccharide is distinct issues in the product on the existing market, because the C-polysaccharide in testing process, counted capsular polysaccharide easily, but the C-polysaccharide does not have immunogenicity, can not stimulate body to produce protection antibody and come sterilization.Consequently sneak into and cause the immune effect of polysaccharide vaccine to be affected in the preparation.
(2), fermented liquid removes the thalline process control
After obtaining to contain the fermented liquid of capsular polysaccharide, the centrifuging of available routine removes thalline wherein.Although centrifugally can remove most of thalline,, still there are some insoluble particulate matter in the solution, comprise thalline, and cell debris, need come further micro-filtration to remove the particulate matter in the solution with microfiltration membrane.Because the development of modern micro-filtration technology through the control of condition, also can directly take the fermented liquid of direct micro-filtration mycetome with microfiltration membrane, come settled solution, to reduce purification step.
(3) control of liquid concentrator acidization
After obtaining to contain the liquid concentrator of capsular polysaccharide; With itself and pollutent; Comprise albumen and nucleic acid; It is numerous and diverse to carry out separation steps, and domestic method adopts and is further purified such as ethanol sedimentation, nucleic acid and protease digestion, organic solvent and phenol extracting and ultrafiltration cleaning step, finally obtains purified polysaccharide.
The present invention has adopted liquid concentrator has been carried out acidifying, protein precipitation and nucleic acid impurity, and this method will reduce traditional multistep widely and remove method suddenly.Different according to the kind of producing bacterium, the perhaps difference of serotype when with pH regulator to the 3.0-5.0 scope of solution, can precipitate thalline, albumen and the nucleic acid of 90%-98% in the fermented liquid.The used acid of souring soln can be phosphoric acid, acetic acid, hydrochloric acid, sulfuric acid, the mixed solution of the different concns of nitric acid or these acid solutions.Acidificatoin time is 10 minutes-24 hours, need decide according to different bacteriums or serotype.The fermented liquid temperature can be 4-37 ℃ during acidifying.Through the control to above condition, principal pollutant such as the thalline in the fermented liquid, albumen and nucleic acid can form deposition fast, and capsular polysaccharide can stably exist in solution and reaches the purpose of separating impurity.
(4), the control of centrifugal and/or microfiltration process
Through ordinary method, remove deposition like centrifugal and/or micro-filtration, in purifying platform program of the present invention, at first with the ferment product after the next centrifugal acidifying of online whizzer, to remove oarse-grained deposition.Centrifuging temperature is 4-25 ℃.Centrifugation time was at 30 minutes-120 minutes.Subsequently, come further to remove the small-particle insolubles in the solution through micro-filtration, the molecular weight ranges of microfiltration membrane is at 0.22 to 1 μ m.Collection contains the clarification filter solution of capsular polysaccharide, abandons the micro-filtration phegma.
(5), the control of ultrafiltration and concentration and filter wash process
The ultra-filtration membrane molecular weight that in the purification technique platform program of the present invention micro-filtrate is carried out ultrafiltration and concentration and clean to adopt can be 5,10,30,50,100 or 500Kd in any, need decide according to bacterial species or particular serotype.The 2-10 that concentrated final solution volume range is an original solution doubly decides according to bacterial species or serotype difference.Adopt any in pure water, damping fluid, salts solution or the acidifying salt soln during ultrafiltration filter wash liquid concentrator process step, needing more, the stability of different serotypes polysaccharide decides.Through above purifying process process, the foreign matter content in the solution progressively reduces, and following table is streptococcus pneumoniae part serotype capsular polysaccharide solution process purifying rear impurity concentration determination result:
Visible from last table, according to the difference of streptococcus pneumoniae serotype,, can remove 90-98% albumen in the polysaccharide soln, and the effect that nucleic acid is removed is more obvious, can reach 97-99% through purification process of the present invention.
Should be noted that purifying capsular polysaccharide technology of the present invention can and be used with other purification process.In general, available purification process of the present invention is removed most of impurity after polysaccharide in the fermented liquid is carried out preliminary purification; In the purifying process in downstream, can adopt other finished purification method, then such as activated carbon filtration; The absorption of phosphorus hydroxyl grey matter rock, chromatography, ethanol sedimentation; And method such as phenol extraction combines to be further purified, and removes residual albumen or contaminant nucleic acid, and obtains refining polysaccharide.
What need further stress is that purification process of the present invention is one step of key of fast purifying bacterial capsule polysaccharide.Can be applied to the part of other purifying capsular polysaccharide technology,, can albumen and nucleic acid content be reduced to below 1.0% through combining other polysaccharide purification method.
More than the present invention is summarized, for further explain and understanding, can read following object lesson.The purpose of describing these examples only is for the present invention will be described, rather than limits the invention in these example scopes.
Embodiment 1:
Purifying capsular polysaccharide from the fermented liquid of streptococcus pneumoniae serotype 19F, the concrete grammar step is following:
Step 1, the fermentation of capsular polysaccharide bacterium: the bacterium that goes out specific capsular polysaccharide in the fermentation cylinder for fermentation that bacteria culture medium is housed;
(1), main seed and the preparation of work seed
Streptococcus pneumoniae serotype 19F is from obtaining from American Type Culture Collecti (American Type Culture Collection); With the bacterial classification inoculation in the freeze-dried semen pipe in 5ml yeast-acid hydrolysis casein nutrient solution; Cultivated 12-24 hour for 36 ℃ ± 2 ℃, when bacterial growth to OD
600When reading is 1.0-1.5, the commentaries on classics of bacterium liquid is seeded in fresh yeast-acid hydrolysis casein nutrient solution of 150ml, under 36 ℃ ± 2 ℃, cultivates 5-10 hour, stop to cultivate to exponential phase of growth, the packing freeze-drying, being stored in 2-8 ℃ is main seed.
The bacterial classification inoculation of the main seed freeze-drying pipe of streptococcus pneumoniae serotype 19F is in 5ml yeast-acid hydrolysis casein nutrient solution, cultivated 12-24 hour for 36 ℃ ± 2 ℃, when bacterial growth to OD
600When reading is 1.0-1.5, the commentaries on classics of bacterium liquid is seeded in fresh yeast-acid hydrolysis casein nutrient solution of 150ml, under 36 ℃ ± 2 ℃, cultivates 5-10 hour to exponential phase of growth, stop to cultivate, the packing freeze-drying is stored in 2-8 ℃ and is the work seed of this serotype.
(2), fermentation using bacteria
Take out the seed pipe from the work seed bank and be seeded to 5ml yeast-acid hydrolysis casein nutrient solution, be cultured to bacterial growth index mid-term at 36 ℃ ± 2 ℃.The commentaries on classics of bacterium liquid is seeded in fresh yeast-acid hydrolysis casein nutrient solution of 150ml; Under 36 ℃ ± 2 ℃; Cultivate 5-10 hour to exponential phase of growth, 50ml bacterium liquid is changeed being seeded in 2L fresh yeast-acid hydrolysis casein nutrient solution, under 36 ℃ ± 2 ℃; Be cultured to mid-term exponential phase of growth and be prepared into fermentation seed liquid, fermentation seed liquid is seeded in 50 liters of fermentor tanks that 30 liters of yeast-acid hydrolysis casein nutrient solution is housed.Keep fermented liquid pH 6.8 ± 0.2 with sodium hydroxide, treat bacterial growth to the index later stage.
Step 2 adopts centrifuging to remove thalline in the step 2 fermented liquid, collects centrifuged supernatant; With online whizzer under 10 ℃, with 8,000～10; Centrifugal 30～50 minutes of 000rpm removes thalline, collects the solution that no insoluble particle contains capsular polysaccharide; Carry out micro-filtration again, carry out micro-filtration, to remove residual cell fragment and insoluble finely ground particle substance wherein with supernatant after the microfiltration membrane spinning; With 0.22 μ m membrane microfiltration fermentation centrifuged supernatant, collect filtered liq, abandon phegma;
Step 3 concentrates the micro-filtrate that contains capsular polysaccharide with filter wash with ultra-filtration membrane;
Step 4 adds acid to liquid concentrator, regulates and contains capsular polysaccharide pH value of solution value 3～5 scopes and come precipitated impurities;
Step 5 adopts centrifuging to remove throw out in the step 4 fermented liquid, collects centrifuged supernatant, with online whizzer under 10 ℃; With 8,000～10, centrifugal 30～50 minutes of 000rpm removes thalline; Collect the solution that no insoluble particle contains capsular polysaccharide, carry out micro-filtration again, carry out micro-filtration, to remove residual cell fragment and insoluble finely ground particle substance wherein with supernatant after the microfiltration membrane spinning; With 0.22 μ m membrane microfiltration fermentation centrifuged supernatant, collect filtered liq, abandon phegma;
Step 6, ultrafiltration filter wash polysaccharide soln obtains the extremely low capsular polysaccharide solution of impure content; Concentrate 10 times of micro-filtrate to original volume with the 30Kd membrane ultrafiltration after, carry out filter wash, remove impurity such as albumen residual in the solution and nucleic acid with pure water; The vacuum-freeze-dry polysaccharide stores down at-70 ℃.
Step 7 adopts prior art that the bacterial capsule polysaccharide is carried out subsequent purification.
Claims (7)
1. the method for fast purifying bacterial capsule polysaccharide may further comprise the steps:
Step 1, the bacterium that goes out to produce capsular polysaccharide in the fermentation cylinder for fermentation that bacteria culture medium is housed;
Thalline in the step 2, centrifugal and/or micro-filtration fermented liquid is collected the solution that no insoluble particle contains capsular polysaccharide;
Step 3 concentrates the micro-filtrate that contains capsular polysaccharide with filter wash with ultra-filtration membrane;
Step 4 adds acid to liquid concentrator, regulates and contains capsular polysaccharide pH value of solution value≤5 scopes and come precipitated impurities;
Step 5, centrifugal and/or micro-filtration is removed the throw out in the solution;
Step 6, ultrafiltration filter wash polysaccharide soln obtains the extremely low capsular polysaccharide solution of impure content.
2. according to the method for the said fast purifying bacterial capsule of claim 1 polysaccharide, it is characterized in that: said bacterium comprises: streptococcus pneumoniae, b type hemophilus influenzae, epidemic meningitis coccus and Corynebacterium diphtheriae.
3. according to the method for the said fast purifying bacterial capsule of claim 1 polysaccharide, it is characterized in that: added acid is any one in phosphoric acid, acetic acid, hydrochloric acid, sulfuric acid or the nitric acid in step 4.
4. according to the method for the said fast purifying bacterial capsule of claim 1 polysaccharide, it is characterized in that: the scope of pH value is 3.0～5.0 in step 4.
5. according to the method for the said fast purifying bacterial capsule of claim 1 polysaccharide, it is characterized in that: the membrane pore size of the used microfiltration membrane of micro-filtration is any one among 0.22,0.45,0.65, the 1.0 μ m.
6. according to the method for the said fast purifying bacterial capsule of claim 1 polysaccharide, it is characterized in that: the used ultra-filtration membrane of ultrafiltration is that molecular weight is any one in 5kD to the 500kD scope.
7. according to the method for the said fast purifying bacterial capsule of claim 1 polysaccharide, it is characterized in that: the used solution of filter wash is pure water, damping fluid or salts solution or their mixed solution.
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