CN107043431B - Purification method of bacterial capsular polysaccharide - Google Patents

Abstract

The invention discloses a purification method of bacterial capsular polysaccharide, which comprises the steps of treating a streptococcus pneumoniae fermentation culture solution by using an inactivating agent, adding acid to adjust the pH value to be less than or equal to 6.5, precipitating impurities, centrifuging and microfiltering to remove precipitates to obtain polysaccharide clarified liquid; performing ultrafiltration liquid exchange and concentration on the polysaccharide clarified solution by using a membrane with the molecular weight cutoff of 10-100 KDa, and performing chromatographic separation on the obtained capsular polysaccharide solution by using composite ion exchange chromatography and hydroxyl phosphate gray salt chromatography to obtain a purified capsular polysaccharide solution; concentrating and ultrafiltering capsular polysaccharide solution, and performing sterile filtration and storage. By combining the compound ion exchange chromatography and the hydroxyl phosphate gray salt chromatography, the protein and nucleic acid impurities can be effectively reduced to be lower than 1%, the quality of the purified polysaccharide product is higher than the standard requirement of European Union pharmacopoeia, and the recovery rate is more than 60%.

Description

Purification method of bacterial capsular polysaccharide

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a purification method of a crude bacterial capsular polysaccharide product.

Background

Streptococcus pneumoniae is a gram-positive bacterium with over 90 serotypes, and most streptococcus pneumoniae bacteria are coated with a layer of capsular polysaccharide. Capsular polysaccharide can escape from the recognition of immune system and prevent phagocytosis of immune cells, thus enabling bacteria to propagate in vivo to cause diseases. Years of research and clinical application prove that the capsular polysaccharide of streptococcus pneumoniae can induce specific antibodies as a vaccine and has good immune protection effect on corresponding streptococcus pneumoniae. The capsular polysaccharides of different serotypes of streptococcus pneumoniae vary in composition and structure and it is therefore often necessary to use combinations of polysaccharides of different serotypes of streptococcus pneumoniae as vaccines to prevent the pathogenicity of major streptococcus pneumoniae.

The streptococcus pneumoniae used for preparing polysaccharide vaccines produces a large amount of metabolites in addition to capsular polysaccharides during fermentation culture. In order to meet the purity of capsular polysaccharide required by vaccine preparation, fermentation broth must be treated through multiple purification steps to remove impurities such as protein, nucleic acid, endotoxin and the like in crude polysaccharide products, so as to avoid side reactions possibly caused by subsequent vaccination.

The establishment of the bacterial capsular polysaccharide purification process has been developed for many years, the traditional bacterial capsular polysaccharide purification process adopts a cold phenol extraction method to remove proteins, the method needs a long centrifugation time, and phenol is used as a reagent with extremely strong corrosion, so that the skin and mucosa of an operator can be corroded, even the central nerve of the operator is inhibited, or the liver and kidney functions are damaged; and the disposal of the waste phenol has a serious threat to environmental protection.

Ethanol precipitation is generally effective in removing protein contaminants from polysaccharides, but it is difficult to achieve the purity requirements for vaccines for injection. In addition, the use of ethanol also causes the following problems: 1) the use of ethanol requires fire protection facilities, which are expensive to design; 2) the amount of ethanol required is huge, each liter of processing material almost needs 4-6L of ethanol, and the waste liquid treatment is very difficult; 3) the treated polysaccharide is difficult to redissolve, and needs freeze-drying and other steps.

Patent US5714354 proposes an improved process for the purification of pneumococcal capsular polysaccharides by ethanol-free precipitation. The method is characterized in that 20 capsular polysaccharides in 23 pneumococcal serotypes are precipitated by Cetyl Trimethyl Ammonium Bromide (CTAB), and then impurities in the polysaccharides are further separated by activated carbon filtration, hydroxyl phosphate gray salt (Hydroxyapatite) chromatography and anion exchange chromatography. The quality of all 23 serotype capsular polysaccharides purified by the method reaches the quality index of polysaccharide vaccines. At present, CTAB has been widely used to selectively precipitate polysaccharides for the purpose of separating proteins and nucleic acids, as in patents CN201510012526 and CN 200910128141. However, the dissociation of the formed CTAB-polysaccharide complex is difficult, the process steps are multiple and complicated, and the recovery rate of the capsular polysaccharide is influenced. Thus, in patent CN200910128141, pure ethanol or ethanol and water mixture is still used to dissolve CTAB-polysaccharide complex.

Lowering the pH of polysaccharide fermentation solutions is also disclosed by patents CN200880012946 and CN201210112391 for selective precipitation of proteins, nucleic acids without seriously affecting polysaccharide yield. The purification method is characterized in that most of pollutants (such as protein, nucleic acid and the like) and capsular polysaccharide in the solution are separated through the acidification step, so that the subsequent process is simplified, and the purification time is shortened. However, we have found that the prepared polysaccharides obtained do not meet pharmacopoeial standards and still require further purification, including adsorption of residual impurities using chromatographic chromatography, and microfiltration, ultrafiltration to obtain acceptable polysaccharides.

In the polysaccharide purification process, polysaccharide (crude sugar) obtained by multi-step ethanol fractional precipitation or CTAB precipitation and acidification treatment for simplifying the purification steps can not reach pharmacopeia standards, and qualified refined sugar can be obtained by further treatment through single or multiple chromatographic steps. C-polysaccharides in solution are removed using Hydrophobic Interaction Chromatography (HIC) or ion exchange chromatography (IEX) during the purification of polysaccharides as reported in patent CN 201180071029. Because some serotype capsular polysaccharide glycosyl groups carry phosphate ions (such as 6A, 6B, 19F and the like), the packing material can be greatly combined with polysaccharide molecules in a chromatography process, so that the polysaccharide needing to be recovered in effluent is remarkably reduced. Patent CN200610048875 uses Sepharose 4FF gel for bacterial polysaccharide purification, but due to the nature of the selected filler, the volume of the treated solution is significantly limited and not suitable for scale-up production. Quyu et al (the New Chinese medicine J2016, volume 25, phase 10) adopt DOE design to establish a Capto Adhere-based pneumonia polysaccharide purification process, but still a small amount of proteins cannot be effectively removed, the chromatographic purification parameter control requirement is very strict, and generally the contents of the proteins and nucleic acids are difficult to reach the standard, so that the purification process is not beneficial to actual production.

Disclosure of Invention

The first purpose of the invention is to provide a purification method for purifying bacterial capsular polysaccharide by chromatographic chromatography, which does not need ethanol precipitation, has simple operation and high recovery efficiency.

A second object of the invention is to provide the use of a combination of complex ion exchange chromatography and hydroxyphosphonate chromatography in a process for the purification of bacterial capsular polysaccharides.

In order to realize the first purpose of the invention, the invention discloses the following technical scheme: the purification method of the bacterial capsular polysaccharide is characterized by comprising the following steps:

(1) treating a streptococcus pneumoniae fermentation culture solution by using an inactivating agent to obtain a bacterial solution containing cell fragments, proteins, nucleic acids and polysaccharides;

(2) adding acid into the bacterial dissolved solution obtained in the step (1) to adjust the pH value to 6.5, precipitating impurities, centrifuging and microfiltering to remove precipitates to obtain polysaccharide clarified liquid;

(3) performing ultrafiltration liquid exchange and concentration on the polysaccharide clarified liquid obtained in the step (2) by using a membrane with the molecular weight cut-off of 10-100 KDa to obtain a capsular polysaccharide solution with most of soluble protein, nucleic acid and large particle impurity removed;

(4) performing chromatographic separation on the capsular polysaccharide solution obtained in the step (3) by using a composite ion exchange chromatography and a hydroxyl phosphate gray salt chromatography in a combined manner to obtain a purified capsular polysaccharide solution;

(5) and (4) concentrating and ultrafiltering the capsular polysaccharide solution obtained in the step (4), and performing sterile filtration and storage.

As a preferable scheme, the acid added in the step (2) is one or more of phosphoric acid, acetic acid, hydrochloric acid, sulfuric acid or nitric acid.

As a preferable mode, the pH value in the step (2) is in the range of 3.0 to 6.5.

As a preferable scheme, the microfiltration membrane used in the step (2) has any one of the membrane pore size of 0.22, 0.45, 0.65 and 1.0 um.

Preferably, the combined use in step (4) is performed by using a complex ion exchange chromatography and then a hydroxyphosphonate chromatography or by using a hydroxyphosphonate chromatography and then a complex ion exchange chromatography.

As a preferred embodiment, the composite type ion exchange chromatography packing is selected from Capto Adhere, TOYOPEARL MX-Trp-650M, Cellufine MAX amino butyl, and CHT is obtained by chromatography of hydroxyl phosphate gray salt^TMCeramicHydroxyapatite。

In order to realize the second purpose of the invention, the invention discloses the following technical scheme: the application of the combined use of composite ion exchange chromatography and hydroxyl phosphate gray salt chromatography in the purification method of bacterial capsular polysaccharide.

The invention may be used to purify capsular polysaccharides from different serotypes containing S.pneumoniae, and is intended to be illustrative and not limiting, and includes serotypes 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.

The invention has the advantages that: by using the compound ion exchange chromatography and the hydroxyl phosphate gray salt chromatography in combination, the protein and nucleic acid impurities can be effectively reduced to be lower than 1%, the quality of the purified polysaccharide product is higher than the standard requirement of European Union pharmacopoeia, and the recovery rate is more than 60%. The method has the advantages of simple and quick process, easy amplification for industrial production, no need of ethanol precipitation, simple operation, high recovery efficiency, low reagent consumption, low contents of protein and nucleic acid in polysaccharide and the like.

Drawings

FIG. 1 ion exchange chromatography (Capto DEAE) pneumococcal polysaccharide 19F chromatogram.

FIG. 2 Complex ion exchange chromatography (Capto adhere) pneumococcal polysaccharide 19F chromatogram.

FIG. 3 a chromatogram of hydroxyl phosphate gray salt chromatography (Hydroxyapatite) pneumococcal polysaccharide 19F.

FIG. 4 SDS-PAGE patterns of protein impurity changes in polysaccharide solutions during chromatographic procedures.

FIG. 5 is a graph showing the trend of protein content in polysaccharide solution during chromatographic process.

FIG. 6 is a graph showing the trend of the change of the nucleic acid content in the polysaccharide solution during the chromatographic process.

FIG. 7 is a graph showing the trend of polysaccharide recovery during chromatographic procedures.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1 preparation of Streptococcus pneumoniae polysaccharide serotypes 1, 3, 4, 7F, 9v, 15B, 19A, 19F, 23F, 33F capsular crude polysaccharide

Dissolving Streptococcus pneumoniae fermentation culture solution with sodium Deoxycholate (DOC) at 4 deg.C for 12-24 hr. And then directly adding acid into the bacterial dissolving solution, adjusting the pH value of the capsular polysaccharide containing dissolving solution to 3-6.5, standing at room temperature for more than 1 hour, and centrifuging to remove impurities such as a dissolving agent, partial protein and nucleic acid, a large amount of cell debris and the like. The acidified solution was centrifuged at 20 ℃ for one hour at a relative centrifugal force of more than 10000g, the supernatant was collected and the precipitate was discarded. And (3) performing ultrafiltration on the obtained supernatant by using a membrane with the molecular cut-off of 50-100kD, performing washing filtration by using pure water to further remove small molecular impurities, and finally concentrating to obtain a crude capsular polysaccharide solution.

TABLE 1 protein and nucleic acid content of different serotypes, production batches and polysaccharide recovery

Example 2.19 analysis of the Effect of serotype F capsular polysaccharide solutions on treatment with different chromatographic Filler

Experimental procedures

Ion exchange chromatography (Capto DEAE, GE): the crude polysaccharide solution obtained after acidification is further ultrafiltered using a membrane with a molecular weight cut-off of 10-100kD, with 5-50mM phosphate buffer at a pH of 6 to 8 until a constant conductance is reached. The Capto DEAE chromatographic column is equilibrated by the same buffer solution, and then the capsular polysaccharide solution after liquid change is loaded on the column, and the flow-through liquid is collected. After the loading was complete, the column was further washed with 1-2 column volumes of 25mM phosphate buffer and the polysaccharide was recovered in the flow-through and wash solutions. The proteins, nucleic acids and polysaccharides bound to the Capto DEAE column were eluted with 1M NaCl.

Complex ion exchange chromatography (Capto adhere, GE): the crude polysaccharide solution obtained after the acidification treatment is further ultrafiltered using a membrane with a molecular weight cut-off of 10-100kD with 5-50mM phosphate buffer, preferably at a salt ion concentration (NaCl, KCl) of 100-. The Capto adhere chromatographic column is equilibrated with the same buffer solution, and then the capsular polysaccharide solution after liquid change is loaded on the column, and the flow-through liquid is collected. After the loading was completed, the column was further washed with 1-2 column volumes of phosphate buffer, buffer containing the same salt ion concentration, and the polysaccharide was recovered in the flow-through and wash solutions. Proteins, nucleic acids and polysaccharides bound to the Captoadhere column were eluted with 0.1M acetic acid.

Hydroxyl phosphate gray salt chromatography (Hydroxyapatite, Bio-Rad): the crude polysaccharide solution obtained after the acidification treatment is further ultrafiltered using a membrane with a molecular weight cut-off of 10-100kD, with 5-50mM phosphate buffer, preferably at a salt ion concentration (NaCl) of 100-. The Hydroxypatite chromatographic column is equilibrated with the same buffer solution, and then the solution of capsular polysaccharide after liquid change is loaded on the column, and the flow-through liquid is collected. After the loading was completed, the column was further washed with 1-2 column volumes of the same salt ion buffer, and the polysaccharide was recovered in the flow-through and wash solutions. Proteins, nucleic acids and polysaccharides bound to the Hydroxypatite column were eluted with 0.5M phosphate buffer.

The crude polysaccharide solution obtained after acidification treatment using a combination of complex ion exchange chromatography (GE) and hydroxyphosphonate chromatography (Hydroxyapatite, Bio-Rad) was further ultrafiltered using a 10-100kD cut-off membrane with 5-50mM phosphate buffer, preferably at a salt ion concentration (NaCl) of 100-. The Hydroxypatite chromatography column, preferably a Capto adhere chromatography column in series, is then equilibrated with the same buffer, and the transudate is collected by loading the column with the changed capsular polysaccharide solution. After the loading was completed, the column was further washed with 1-2 column volumes of the same salt ion buffer, and the polysaccharide was recovered in the flow-through and wash solutions. Proteins, nucleic acids and polysaccharides bound on the Captoadhere column and on the Hydroxyaptite column were eluted separately. In addition, after the concentration of the salt ions of the solution containing capsular polysaccharide recovered after the Capto adhere chromatography is adjusted (or not adjusted) by the ultrafiltration exchange solution, the solution continues to go up a Hydroxyapatite chromatography column, and the polysaccharide-containing flow-through solution is collected. On the contrary, the acidified crude sugar solution may be passed through a hydro xyaptite chromatographic column and then a Capto adhere chromatographic column after the buffer solution salt concentration is adjusted.

Analysis of results

As can be seen from the chromatogram of pneumococcal polysaccharide serotype 19F ion exchange chromatography (Capto DEAE) in fig. 1, the polysaccharide peak in the flow-through of Capto DEAE chromatography is significantly lower than that of Capto Adhere chromatography (fig. 2) and that of hydroxypart chromatography (fig. 3), and it is presumed that a large portion of capsular polysaccharide is adsorbed onto the Capto DEAE packing, resulting in a decrease in the polysaccharide concentration in the flow-through. Because, under alkaline to neutral pH conditions, serotype 19F capsular polysaccharide is negatively charged due to the phosphate ion on the glycosyl group and is adsorbed onto Capto DEAE. This indicates that Capto DEAE is not suitable for the purification of this type of polysaccharide of serotype containing phosphate ions.

The Capto Adhere chromatography was reported to be used for the development of the pneumococcal polysaccharide purification process (vol 25, 10 of the new journal of china 2016), but as can be seen from the SDS-PAGE pattern of the protein in the polysaccharide solution in fig. 4, a part of the protein remained in the polysaccharide solution after the Capto Adhere chromatography (the content is about 10-20%). Whereas the residual protein band in the polysaccharide solution after Hydroxyaptite chromatography was less than that of Capto Adhere chromatography. After two different chromatographic methods are used for treatment, the sizes of the residual protein bands are also obviously different, which shows that the two chromatographic fillers have certain difference on the adsorption of the protein. Thus, after combined use of Capto Adhere and hydro xypatite chromatography, almost no protein residue was seen in the polysaccharide solution. The results show that the combined use of two chromatographic steps of composite ion exchange chromatography and hydroxyl phosphate gray salt chromatography can be effectively used for purifying the bacterial capsular polysaccharide.

TABLE 2.19 results of different chromatographic analyses of capsular polysaccharides

Purification method	Protein content%	Nucleic acid content%	Polysaccharide recovery rate%
				Capto DEAE	9.6	0.3	15.4
Capto Adhere	14.3	0.6	85
				Hydroxyapatite	6.7	0.5	78.1
Capto Adhere，Hydroxyapatite	0.5	0.1	63.6

Example 3 preparation of Streptococcus pneumoniae polysaccharide serotypes 1, 3, 4, 7F, 9v, 15B, 19A, 19F, 23F, 33F

The crude polysaccharide solution obtained after the acidification treatment is further ultrafiltered using a membrane with a molecular weight cut-off of 10-100kD, with 5-50mM phosphate buffer, preferably at a salt ion concentration (NaCl) of 100-. The Hydroxypatite chromatography column, preferably a Capto adhere chromatography column in series, is then equilibrated with the same buffer, and the transudate is collected by loading the column with the changed capsular polysaccharide solution. After the loading was completed, the column was further washed with 1-2 column volumes of the same salt ion buffer, and the polysaccharide was recovered in the flow-through and wash solutions.

TABLE 3 protein and nucleic acid content and polysaccharide recovery for different serotypes after Capto Adhere and Hydroxypatite chromatography

The invention relates to a method for purifying streptococcus pneumoniae polysaccharide based on chromatography, which can avoid the non-specific adsorption of serum polysaccharide (such as 6A, 6B, 19F and the like) with phosphate ion groups by common ion exchange chromatography by using compound ion exchange chromatography. The combination of hydroxyl phosphate gray salt chromatography is favorable for further reducing impurities such as protein in the polysaccharide solution. Capsular polysaccharides prepared by the present process have about 60 to 80% recovery, with less than 1% protein and less than 1% nucleic acid. The process has been carried out on a research, pilot scale. This method can save 50-90% of time and reduce 90% of cost for purifying polysaccharides compared to methods based on CTAB or alcohol precipitation.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (2)

1. The purification method of the bacterial capsular polysaccharide is characterized by comprising the following steps:

(1) treating a streptococcus pneumoniae fermentation culture solution by using an inactivating agent to obtain a bacterial solution containing cell fragments, proteins, nucleic acids and polysaccharides;

(2) adding acid into the bacterial dissolved solution obtained in the step (1) to adjust the pH value to 3.0-6.5, precipitating impurities, centrifuging and microfiltering to remove precipitates to obtain polysaccharide clarified liquid, wherein the acid is one or more of phosphoric acid, acetic acid, hydrochloric acid, sulfuric acid or nitric acid;

(3) performing ultrafiltration liquid exchange and concentration on the polysaccharide clarified liquid obtained in the step (2) by using a membrane with the molecular weight cut-off of 10-100 KDa to obtain a capsular polysaccharide solution with most of soluble protein, nucleic acid and large particle impurity removed;

(4) performing chromatographic separation on the capsular polysaccharide solution obtained in the step (3) by using a composite ion exchange chromatography and a hydroxyl phosphate gray salt chromatography in a combined manner to obtain a purified capsular polysaccharide solution; the combined use refers to firstly using compound ion exchange chromatography and then using hydroxyl phosphate gray salt chromatography or firstly using hydroxyl phosphate gray salt chromatography and then using compound ion exchange chromatography; the composite ion exchange chromatography filler is selected from Capto Adhere, TOYOPEARL MX-Trp-650M and Cellufine MAX amino butyl, and the hydroxyl phosphate gray salt chromatography is CHT (fragment of human serum) chromatography;

(5) and (4) concentrating and ultrafiltering the capsular polysaccharide solution obtained in the step (4), and performing sterile filtration and storage.

2. The purification process of bacterial capsular polysaccharide according to claim 1 wherein the microfiltration membrane used in step (2) has a membrane pore size of any one of 0.22, 0.45, 0.65, 1.0 um.

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