Method for purifying capsular polysaccharides

Abstract

The present invention relates broadly to an improved process for purifying capsular polysaccharides. In particular, the present invention relates to a method for purifying capsular polysaccharides from Streptococcus pneumoniae, as well as other similar related capsular polysaccharides produced by Gram-negative and Gram-positive microorganisms. The present invention relates to a method for purifying capsular polysaccharides from Streptococcus pneumoniae, as well as other similar related capsular polysaccharides produced by Gram-negative and Gram-positive microorganisms. More specifically, the invention relates to passing a crude capsular polysaccharide solution through a reinforced cellulose membrane having a quaternary ammonium, further contacting the solution with silicon dioxide (SiO2), and isolating the capsular polysaccharide in pure form. The purified capsular polysaccharide can be used to produce vaccines containing only the polysaccharide or containing a polysaccharide conjugated to a protein.

Description

Technical Field

[0001] The present invention broadly relates to an improved method for purifying capsular polysaccharides. Specifically, the present invention relates to a method for purifying capsular polysaccharides from Streptococcus pneumoniae and other similar related capsular polysaccharides produced by Gram-negative and Gram-positive microorganisms. More specifically, the present invention relates to passing a crude capsular polysaccharide solution through an enhanced cellulose membrane having a quaternary ammonium, further contacting the solution with silica (SiO2), and isolating the capsular polysaccharide in pure form. The purified capsular polysaccharide can be used to produce vaccines containing only the polysaccharide or the polysaccharide conjugated to a protein. Background Art

[0002] Each stage of the vaccine manufacturing process is crucial to ensure its safety for human use. Polysaccharides are carbohydrates used in a variety of industrial applications such as thickeners, gelling agents, emulsifiers, and delivery systems for many commercial products. Capsular polysaccharides present on the surface of microbial cells can be used as components for immunization. After immunization with purified capsular polysaccharides in a formulated composition, it prevents pathogenic organisms such as Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzaetype b, and Salmonella typhi by inducing the corresponding immune response. This makes them an important part of vaccine design.

[0003] Capsular polysaccharides have also been shown to be useful in eliciting an immune response, especially when conjugated to a carrier protein. Conjugate vaccines result in enhanced immunogenic responses including in children, immunodeficient individuals, and the elderly population. Polysaccharides conjugated to proteins such as CRM197, tetanus toxoid, diphtheria toxoid, and other surface proteins have been well validated and are highly immunogenic.

[0004] All immunogenic or vaccine preparations approved for human use require highly purified forms of capsular polysaccharides. Capsular polysaccharides are present on the outer surface of bacterial cells. During the process of separating capsular polysaccharides from the cells, cellular components such as nucleic acids, proteins, cell walls, etc. are released. In addition to the biosynthetic products produced during fermentation, nutrients in the culture medium can also be a cause of contaminants. The separation / purification process of capsular polysaccharides involves multiple steps including chromatographic separation, filtration, treatment with detergents, solvents, enzymes to hydrolyze nucleic acids, proteins, polysaccharides, etc.

[0005] The following references disclose various methods for removing proteins and other impurities from capsular polysaccharides.

[0006] Canadian Patent No. CA1206905 describes a method using toxic organic solvents such as phenol, butanol, toluene, and chloroform, and using the detergent Cetavlon (CTAB).

[0007] European Patent No. EP0497525 discloses methods related to hot hydrolysis and hydrolysis of samples with sodium acetate.

[0008] US Patent No. 5,847,112 mentions a method of precipitating polysaccharides using multiple isopropanol precipitations and Cetavlon precipitation.

[0009] US Patent Publication No. 20060228380 discloses a method including using Cetavlon to precipitate polysaccharides, using a carbon filter to remove nucleic acids, and using potassium iodide to precipitate Cetavlon.

[0010] WO Publication No. 2006 / 082527A2 discloses a purification process for Streptococcus agalactiae capsular polysaccharide, in which the sugar is first treated with an aqueous mixture of alcohol and calcium salt, and then precipitated with a cationic detergent.

[0011] IN 1572 / MUM / 2010 discloses a purification process for removing protein contaminants, which involves treating a nuclease-treated polysaccharide solution with a mixture of detergent and brine; then centrifugation, diafiltration, and chromatography are carried out.

[0012] WO Publication No. 2012 / 127485Al discloses a method for purifying Streptococcus pneumoniae polysaccharide without using alcohol and CTAB, which utilizes chromatographic separation of capsular polysaccharide (PnP) based on net surface charge differences.

[0013] Referring to the prior art, it has been observed that removing process impurities from capsular polysaccharides is very challenging because both carry negative charges.

[0014] The processes disclosed in the above prior art involve multiple complex steps, which will have an adverse impact on yield, quality, stability, processing time, and process operation. In addition, these processes are costly to operate, require high skills to carry out, and require a large amount of time to reduce the impurity level.

[0015] Furthermore, if the initial load is high and produced in the form of aggregates, the processes disclosed in the prior art cannot efficiently remove process impurities in bacterial capsular polysaccharides. Therefore, there is a need to improve the method for removing impurities from complex cell lysates. Through continuous efforts, the inventors have found that using enhanced cellulose membranes and silica with quaternary ammonium ligands will effectively remove impurities. In order to bind highly negatively charged DNA, endotoxins, and host cell proteins to the enhanced cellulose membrane with quaternary ammonium as a ligand, an optimized pH range is adopted.

[0016] The method developed by the inventors is simple, efficient, and can be easily scaled up.

[0017] Objects of the Invention

[0018] An important object of the present invention is to provide an improved method for purifying capsular polysaccharides from Streptococcus pneumoniae and other similar related capsular polysaccharides produced by Gram-negative and Gram-positive microorganisms, and to provide purified capsular polysaccharides that meet the requirements of the pharmacopoeia.

[0019] Another object of the present invention is to provide a simple and cost-effective method for purifying capsular polysaccharides without using complex and expensive chromatographic methods and toxic reagents such as phenol.

[0020] Furthermore, another important object of the present invention is to provide a method for robustly purifying capsular polysaccharides on a commercial scale, which requires low material costs and does not require specialized facilities or handling of hazardous materials. Summary of the Invention

[0021] The present invention provides a method for purifying capsular polysaccharides, wherein the method comprises the following steps:

[0022] a. Culturing bacterial cells under standard growth conditions to obtain a fermentation broth;

[0023] b. Lysing the bacterial cells in the fermentation broth obtained in step (a) to obtain a bacterial cell lysate containing capsular polysaccharides, proteins, nucleic acids, cell wall components, and other impurities;

[0024] c. Controlling the size of the bacterial cell lysate in step (b) using a homogenizer to obtain a size-controlled crude capsular polysaccharide solution;

[0025] d. Passing the size-controlled crude capsular polysaccharide obtained in step (c) through an enhanced cellulose membrane having quaternary ammonium as a ligand; and

[0026] e. Contacting the solution obtained after passing through the enhanced cellulose membrane having quaternary ammonium in step (d) with SiO2 and separating the capsular polysaccharides in pure form.

[0027] In one embodiment, the method of the present invention does not require the use of complex and expensive chromatographic methods and toxic reagents such as phenol to purify capsular polysaccharides.

[0028] In another embodiment of the present invention, the capsular polysaccharides are selected from the Streptococcus pneumoniae serotypes of serotype 1 and serotype 5.

[0029] In yet another embodiment, the purification method of the present invention is an alcohol-free process and does not use CTAB or other irritating chemicals. Detailed Description of the Invention

[0030] The embodiments described herein can be more easily understood with reference to the following detailed description and examples. The elements and methods described herein are only for illustrating the principles of the present invention and are not limited to the specific embodiments set forth in the detailed description and examples. Those skilled in the art can easily make various modifications and adjustments without departing from the spirit and scope of the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this method belongs. Although any methods, devices, kits, reagents or compositions similar or equivalent to those described herein can also be used to practice or test these methods, only representative exemplary methods and compositions are described herein.

[0032] It should be understood that, for clarity, certain method features described in different embodiments can also be provided in combination in a single embodiment. Conversely, for brevity, the various method and composition features described in a single embodiment can also be provided separately or in any suitable sub-combination.

[0033] Note that, unless the context clearly dictates otherwise, the singular forms "a / an" and "the" as used herein and in the appended claims include plural referents. It should also be noted that the claims may exclude any optional elements when being drafted. Thus, this statement is intended to serve as antecedent basis for the use of exclusive terms such as "only", "solely", etc. when reciting claim elements or using "negative" limitations. The term "sized / sizing" as used herein refers to reducing the size of natural polysaccharides by various methods.

[0034] "Lysis agent" refers to any reagent that helps in the breakdown of cell walls, such as detergents. As used herein, the term "detergent" refers to any anionic or cationic detergent that can induce lysis of bacterial cells.

[0035] The terms "exposed" or "contacted" refer to incubating the capsular polysaccharide preparation with other components, thereby treating the sample to remove impurities and thus producing a pure polysaccharide.

[0036] For those skilled in the art, it will be apparent after reading this disclosure that each embodiment described and illustrated herein has separate components and features, and these components and features can be easily separated from or combined with the features of any one of the other embodiments without departing from the scope or spirit of this method. Any of the described methods can be carried out in the order of the described events or any other logically feasible order.

[0037] Unless otherwise explicitly stated, the practice of the present invention will employ conventional methods of virology, immunology, microbiology, and molecular biology within the skill of the art. These techniques are well explained in the existing literature.

[0038] Removing process impurities from capsular polysaccharides is a challenging step because both the impurities and the capsular polysaccharides are negatively charged. The isoelectric point is the pH at which a biomolecule has no net charge. Thus, in the present invention, an optimized pH range is employed to bind highly negatively charged DNA, endotoxins, and host cell proteins to an enhanced cellulose membrane having quaternary ammonium as a ligand.

[0039] The present invention provides an improved method for purifying capsular polysaccharides, wherein the source of the capsular polysaccharides is Gram-negative and Gram-positive microorganisms. The improved method involves passing a crude capsular polysaccharide solution through an enhanced cellulose membrane having quaternary ammonium and further contacting the solution with SiO2. After the resulting solution is passed through the enhanced cellulose membrane having quaternary ammonium and further exposed to SiO2, the polysaccharide is enriched and one or more impurities (such as proteins, nucleic acids, cell wall polysaccharides, and other cell-derived substances) are reduced.

[0040] The capsular polysaccharides obtained according to the present invention are in a (substantially) pure form.

[0041] An important aspect of the present invention relates to removing impurities from a complex bacterial cell lysate containing crude capsular polysaccharides. The method involves using an enhanced cellulose membrane having a quaternary ammonium ligand and silica, thereby effectively removing impurities. Importantly, it has been observed that process impurities are significantly reduced and no loss of capsular polysaccharides occurs.

[0042] In another aspect of the present invention, the purified capsular polysaccharides meet all release test specifications of the World Health Organization - Technical Report Series - 927. In addition, the WHO specifications for the purified capsular polysaccharides are as follows:

[0043] Protein content : Usually not more than 2% of the dry weight of the polysaccharide, depending on the polysaccharide serotype. For serotype 1, it is not more than 2%, and for serotype 5, it is not more than 3%.

[0044] Nucleic acid content : Not more than 2% of the dry weight of the polysaccharide.

[0045] Endotoxin content : Less than 0.5 IU / μg of polysaccharide.

[0046] Some important embodiments of the present invention are as follows:

[0047] Important embodiments of the present invention relate to a method for purifying crude capsular polysaccharides, wherein the method comprises the following steps:

[0048] i. Pass a bacterial cell lysate containing capsular polysaccharide, protein, nucleic acid, cell wall components, and other impurities through an enhanced cellulose membrane having a quaternary ammonium as a ligand; and

[0049] ii. Contact the solution obtained in step (i) after passing through the enhanced cellulose membrane having a quaternary ammonium with SiO2, and isolate the capsular polysaccharide in pure form.

[0050] A preferred embodiment of the present invention relates to a method for purifying a crude capsular polysaccharide, wherein the method comprises the following steps:

[0051] a. Cultivate bacterial cells under standard growth conditions to obtain a fermentation broth;

[0052] b. Lyse the bacterial cells in the fermentation broth obtained in step (a) to obtain a bacterial cell lysate containing capsular polysaccharide, protein, nucleic acid, cell wall components, and other impurities;

[0053] c. Subject the bacterial cell lysate in step (b) to size control using a homogenizer to obtain a size-controlled crude capsular polysaccharide solution;

[0054] d. Pass the size-controlled crude capsular polysaccharide solution obtained in step (c) through an enhanced cellulose membrane having a quaternary ammonium as a ligand; and

[0055] e. Contact the solution obtained in step (d) after passing through the enhanced cellulose membrane having a quaternary ammonium with SiO2, and isolate the capsular polysaccharide in pure form.

[0056] In one embodiment, the capsular polysaccharide of the present invention is isolated from bacteria.

[0057] In one embodiment of the present invention, the bacteria are Gram-positive bacteria. On the one hand, the bacteria are selected from, but not limited to, Streptococcus, Staphylococcus, Enterococci, Bacillus, Corynebacterium, Listeria, Erysipelothrix, or Clostridium. On the other hand, the bacteria are selected from, but not limited to, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Group C and Group G streptococci, or Staphylococcus aureus.

[0058] In another embodiment of the present invention, the bacterium is a Gram-negative bacterium. On the one hand, the bacterium is selected from, but not limited to, Haemophilus, Neisseria or Klebsiella. On the other hand, the bacterium is selected from, but not limited to, Haemophilus influenzae, Neisseria meningitidis or Klebsiella pneumoniae.

[0059] In another important embodiment of the present invention, the bacterium is selected from, but not limited to, Streptococcus, Staphylococcus, Enterococcus, Bacillus, Corynebacterium, Listeria, Erysipelothrix, Clostridium, Haemophilus, Neisseria or Klebsiella.

[0060] In yet another embodiment of the present invention, the bacterium is selected from, but not limited to, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Group C and Group G streptococci, Staphylococcus aureus, Haemophilus influenzae, Neisseria meningitidis or Klebsiella pneumoniae.

[0061] In another embodiment of the present invention, the capsular polysaccharide is isolated from, but not limited to, Streptococcus pneumoniae serotypes selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10A, 10B, 10C, 10D, 10F, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F, 16A, 16F, 17A, 17F, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32A, 32F, 33A, 33C, 33D, 33E, 33F, 33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A and 48.

[0062] In an exemplary embodiment of the present invention, the capsular polysaccharide is isolated from Streptococcus pneumoniae serotypes selected from serotypes 1 and 5.

[0063] In another embodiment of the present invention, the improved purification method is an alcohol-free process and does not use CTAB or other irritating chemicals.

[0064] In one embodiment of the present invention, capsular polysaccharide is produced by growing bacteria in a culture medium (e.g., a solid or preferably a liquid culture medium).

[0065] In another embodiment of the present invention, the starting material for the method of the present invention is a bacterial culture, and preferably a liquid bacterial culture (e.g., a fermentation broth).

[0066] In an exemplary embodiment, the bacterial culture is grown in a liquid MH medium. The bacterial culture is grown in a fermenter with the following controlled parameters: the temperature is in the range of 35 ± 2 °C, the pH is in the range of 7.1 ± 0.3, and the back pressure NMT is in the range of 0.2 to 1.0 Bar. The optical density is monitored every 30 to 60 minutes. In addition, when the culture enters the stationary phase or the decline phase, the fermentation is stopped. Thereafter, capsular polysaccharide is prepared by treating the bacterial cells using various methods such as, but not limited to, cell lysis, centrifugation, depth filtration, concentration / diafiltration operations, and precipitation steps, etc.

[0067] In one embodiment, the fermentation broth is inactivated and lysed to obtain a bacterial cell lysate. Cell debris is removed from the inactivated fermentation broth using a continuous centrifuge. The supernatant is subjected to depth filtration and concentrated and diafiltered using a phosphate buffer solution, and optionally ultrafiltered. The solution obtained after pretreatment of the bacterial cell lysate is called a crude polysaccharide solution.

[0068] In one embodiment, a lysing agent is used to lyse the fermentation broth.

[0069] In a preferred embodiment of the present invention, the lysing agent is selected from, but not limited to, sodium deoxycholate (DOC), N-lauroylsarcosine (NLS), sodium chenodeoxycholate, and saponins.

[0070] In another embodiment of the present invention, the fermentation broth is treated with a 0.1% w / v to 0.5% w / v DOC solution to lyse the cells in the fermentation broth.

[0071] In one embodiment, size control of the capsular polysaccharide in the crude capsular polysaccharide solution or the bacterial cell lysate is performed before passing through an enhanced cellulose membrane having an ammonium salt as a ligand. The solution obtained after size control of the crude polysaccharide solution or the bacterial cell lysate is called a size-controlled crude polysaccharide solution.

[0072] In another embodiment, in order to avoid cross-linking of process impurities and product impurities and to make the capsular polysaccharide tough for size control, before the purification process, a size control step of introducing a chemical size control method or treating with a high-pressure homogenizer is used to remove high-molecular-weight product aggregates in the bacterial cell lysate containing capsular polysaccharide.

[0073] In an exemplary embodiment, the capsular polysaccharide is reduced in size by various mechanical methods known in the art, such as high-pressure techniques, such as microfluidization, Emulsiflex TM , high-pressure homogenization, ultrasonication or Gaulin homogenization. Homogenization is achieved by pumping the process stream through a flow path small enough to achieve a high shear rate. The shear rate is increased by applying a greater homogenization pressure and the exposure time is increased by recirculating the feed stream through the homogenizer.

[0074] In one embodiment, the homogenization is carried out at a pressure of at least 500 bar until a polysaccharide of the desired size is obtained.

[0075] In one embodiment of the present invention, the homogenization is preferably carried out at a pressure of 500 to 1000 bar and a flow rate of 50 L / hr to 200 L / hr.

[0076] In one embodiment, the molecular weight of the size-controlled polysaccharide ranges from 50 kDa to 1000 kDa.

[0077] In one embodiment, the molecular weight of the size-controlled polysaccharide ranges from 400 kDa to 700 kDa.

[0078] In a preferred embodiment, the molecular weight of the size-controlled polysaccharide ranges from about 150 kDa to 350 kDa.

[0079] In one embodiment, the size-controlled crude capsular polysaccharide solution is passed through an enhanced cellulose membrane having a quaternary ammonium ligand.

[0080] In one embodiment, the enhanced cellulose membrane having a quaternary ammonium as a ligand is used in any form (such as a matrix or a filter capsule).

[0081] In one embodiment, the enhanced cellulose membrane having a quaternary ammonium ligand is first washed with an alkali and then equilibrated with a buffer before use.

[0082] In one embodiment, the membrane is disinfected with 1N NaOH for at least 30 minutes.

[0083] In another embodiment of the present invention, the enhanced cellulose membrane having a quaternary ammonium ligand is disinfected with 0.5M NaOH and then equilibrated with a phosphate buffered saline solution.

[0084] In another embodiment of the present invention, the enhanced cellulose membrane having a quaternary ammonium ligand is equilibrated with a buffer until the pH reaches 6.5 to 7.5 and the conductivity reaches 2 mS / cm to 4 mS / cm.

[0085] In yet another embodiment of the present invention, the enhanced cellulose membrane having quaternary ammonium ligands is equilibrated with a buffer until the pH reaches 6.5 to 6.8 and the conductivity reaches 3.2 ± 0.3 mS / cm.

[0086] In one embodiment, prior to passing the size-controlled crude capsular polysaccharide solution through the enhanced cellulose membrane having quaternary ammonium ligands, the pH of the solution is adjusted to a range of about 6.0 to 10.0.

[0087] In a preferred embodiment, prior to passing the size-controlled crude capsular polysaccharide solution through the enhanced cellulose membrane having quaternary ammonium ligands, the pH of the solution is adjusted to a range of about 6.5 to 6.8.

[0088] In one embodiment of the present invention, prior to passing the size-controlled crude capsular polysaccharide solution through the enhanced cellulose membrane having quaternary ammonium ligands, the conductivity of the solution is adjusted to a range of about 1.0 mS / cm to 5.0 mS / cm.

[0089] In a preferred embodiment of the present invention, prior to passing the size-controlled crude capsular polysaccharide solution through the enhanced cellulose membrane having quaternary ammonium ligands, the conductivity of the solution is adjusted to a range of about 2.8 mS / cm to 3.2 mS / cm.

[0090] In one embodiment of the present invention, the conductivity of the size-controlled crude capsular polysaccharide solution is adjusted using a 0.5 to 5 M NaCl or 0.5 to 5 M KCl solution.

[0091] In one embodiment of the present invention, the size-controlled crude capsular polysaccharide solution passes through the membrane at a flow rate of about 2 L / min to 10 L / min.

[0092] In a preferred embodiment of the present invention, the size-controlled crude capsular polysaccharide solution passes through the membrane at a flow rate of about 3 L / min to 5 L / min.

[0093] In another embodiment of the present invention, the size-controlled crude capsular polysaccharide solution passes through the enhanced cellulose membrane at a flow rate of 2 to 5 L / min.

[0094] In one embodiment of the present invention, the flow-through solution obtained after passing the size-controlled crude capsular polysaccharide solution through the enhanced cellulose membrane having quaternary ammonium ligands is exposed to or contacted with SiO2.

[0095] In one embodiment, after the contact, the residual SiO2 is removed by centrifugation.

[0096] In one embodiment of the present invention, the solution obtained by passing a size-controlled crude capsular polysaccharide solution through an enhanced cellulose membrane with a quaternary ammonium ligand is then exposed to or contacted with SiO2 without any intermediate treatment or purification process.

[0097] In one embodiment, the SiO2 used has different forms / sizes, for example, fine particles in the range of 0.01 μm to 200 μm.

[0098] In another embodiment, the SiO2 used has different forms / sizes, for example, fine particles preferably in the range of 3 μm to 40 μm.

[0099] In a preferred embodiment, the SiO2 particle size is in the range of 10 μm to 80 μm, more preferably in the range of 20 μm to 60 μm.

[0100] In one embodiment of the present invention, the amount of SiO2 can be 0.5% (w / v) to 20% (w / v).

[0101] In a preferred embodiment, the amount of SiO2 ranges from 5% (w / v) to 7% (w / v) (50 g / L to 70 g / L).

[0102] In one embodiment, the SiO2 used is optionally prepared by heating to above 60 °C for at least 1 hour and cooling before use.

[0103] In another embodiment, the SiO2 used can be pyrogenic or depyrogenic.

[0104] In another embodiment, while contacting with SiO2, the pH value of the solution obtained by passing a size-controlled crude capsular polysaccharide solution through an enhanced cellulose membrane with a quaternary ammonium ligand is maintained within the range from the acidic region to the alkaline region, preferably from 3.0 to 9.0.

[0105] In another embodiment, the pH value of the solution obtained by passing a size-controlled crude capsular polysaccharide solution through an enhanced cellulose membrane with a quaternary ammonium ligand is adjusted with acids (such as acetic acid, phosphoric acid, formic acid, hydrochloric acid, etc.) and bases (such as sodium hydroxide, potassium hydroxide or ammonium hydroxide, etc.).

[0106] In a preferred embodiment, while contacting with SiO2, the pH of the solution obtained by passing a size-controlled crude capsular polysaccharide solution through an enhanced cellulose membrane with a quaternary ammonium ligand is maintained within the range of 5.0 to 7.0.

[0107] In one embodiment, the contact or exposure of the solution obtained by passing a size-controlled crude capsular polysaccharide solution through an enhanced cellulose membrane with a quaternary ammonium ligand to SiO2 is carried out at a temperature of 15 °C to 60 °C for 10 minutes to 16 hours.

[0108] In another embodiment, the contact or exposure of the solution obtained by passing a size-controlled crude capsular polysaccharide solution through an enhanced cellulose membrane with a quaternary ammonium ligand to SiO2 is carried out at room temperature (22 ± 4 °C) for 1 hour to 2 hours.

[0109] In yet another embodiment, the contact or exposure of the solution obtained by passing a size-controlled crude capsular polysaccharide solution through an enhanced cellulose membrane with a quaternary ammonium ligand to SiO2 is carried out at a temperature of 50 ± 5 °C for 60 to 80 minutes.

[0110] In a preferred embodiment of the present invention, the contact with SiO2 is preferably carried out under stirring at a temperature of 15 °C to 25 °C for 60 minutes to 80 minutes.

[0111] In another embodiment of the present invention, SiO2 is removed by centrifugation with a force of 12000 to 14000 g.

[0112] In another embodiment of the present invention, a commercially common brand of SiO2 (silicon dioxide) is used

[0113] In another embodiment, the present invention relates to treating a purified capsular polysaccharide solution with activated carbon to remove pigment impurities. This treatment is carried out after exposure to SiO2.

[0114] In one embodiment, the present invention provides a method for purifying capsular polysaccharides from Streptococcus pneumoniae serotypes 1 and 5, wherein the method comprises the following steps:

[0115] a. Culturing bacterial cells under standard growth conditions to obtain a fermentation broth;

[0116] b. Lysing the bacterial cells in the fermentation broth obtained in step (a) to obtain a bacterial cell lysate containing capsular polysaccharides, proteins, nucleic acids, cell wall components and other impurities;

[0117] c. Controlling the size of the bacterial cell lysate in step (b) with a homogenizer to obtain a size-controlled crude capsular polysaccharide solution;

[0118] d. Passing the size-controlled crude capsular polysaccharide solution obtained in step (c) through an enhanced cellulose membrane with quaternary ammonium as a ligand; and

[0119] e. Contact the solution obtained after passing through the enhanced cellulose membrane with quaternary ammonium as a ligand in step (d) with SiO2, and separate the capsular polysaccharide in pure form.

[0120] In one embodiment, the present invention provides a method for purifying a capsular polysaccharide, wherein the method comprises the following steps:

[0121] a. Cultivate bacterial cells under standard growth conditions to obtain a fermentation broth;

[0122] b. Lyse the bacterial cells in the fermentation broth obtained in step (a) to obtain a bacterial cell lysate containing capsular polysaccharide, protein, nucleic acid, cell wall components and other impurities;

[0123] c. Subject the lysate to a preliminary purification process including centrifugation, depth filtration, and diafiltration to obtain a preliminarily treated crude polysaccharide solution;

[0124] d. Control the size of the preliminarily treated crude polysaccharide solution in step (c) with a homogenizer to obtain a size-controlled crude capsular polysaccharide solution;

[0125] e. Pass the size-controlled crude capsular polysaccharide solution obtained in step (d) through an enhanced cellulose membrane with quaternary ammonium as a ligand; then

[0126] f. Contact the solution obtained after passing through the enhanced cellulose membrane with quaternary ammonium as a ligand in step (e) with SiO2, and separate the capsular polysaccharide in pure form.

[0127] In one embodiment, the present invention provides a method for purifying the capsular polysaccharides of Streptococcus pneumoniae serotypes 1 and 5, wherein the method comprises the following steps:

[0128] a. Cultivate bacterial cells under standard growth conditions to obtain a fermentation broth;

[0129] b. Lyse the bacterial cells in the fermentation broth obtained in step (a) to obtain a bacterial cell lysate containing capsular polysaccharide, protein, nucleic acid, cell wall components and other impurities;

[0130] c. Subject the lysate to a preliminary purification process including centrifugation, depth filtration, and diafiltration to obtain a preliminarily treated crude polysaccharide solution;

[0131] d. Control the size of the preliminarily treated crude polysaccharide solution in step (c) with a homogenizer to obtain a size-controlled crude capsular polysaccharide solution;

[0132] e. Pass the size-controlled crude capsular polysaccharide solution obtained in step (d) through an enhanced cellulose membrane with quaternary ammonium as a ligand; then

[0133] f. Contact the solution obtained after passing through the enhanced cellulose membrane with quaternary ammonium as a ligand in step (e) with SiO2, and separate the capsular polysaccharide in pure form.

[0134] In one embodiment, the filtration in step (c) above is carried out using a 100KDa MWCO cassette for phosphate buffer.

[0135] In one embodiment, the purified capsular polysaccharide solution obtained after contact with SiO2 can be further clarified using a depth filter, a carbon filter, and / or a 0.45 micron filter to obtain a filtered polysaccharide solution.

[0136] In one embodiment, the purified capsular polysaccharide solution can be further passed through an enhanced cellulose membrane with quaternary ammonium as a ligand to remove process impurities, obtaining a super-purified capsular polysaccharide solution.

[0137] In one embodiment, the present invention provides a method for purifying capsular polysaccharide, wherein the method comprises the following steps:

[0138] a. Culturing bacterial cells under standard growth conditions to obtain a fermentation broth;

[0139] b. Lysing the bacterial cells in the fermentation broth obtained in step (a) to obtain a bacterial cell lysate containing capsular polysaccharide, protein, nucleic acid, cell wall components, and other impurities;

[0140] c. Subjecting the lysate to a preliminary purification process including centrifugation, depth filtration, and diafiltration to obtain a preliminarily treated crude polysaccharide solution;

[0141] d. Controlling the size of the preliminarily treated crude polysaccharide solution in step (c) using a homogenizer to obtain a size-controlled crude capsular polysaccharide solution;

[0142] e. Passing the size-controlled crude capsular polysaccharide solution obtained in step (d) through an enhanced cellulose membrane with quaternary ammonium as a ligand; then

[0143] f. Contacting the solution obtained after passing through the enhanced cellulose membrane with quaternary ammonium as a ligand in step (e) with SiO2 to obtain a purified capsular polysaccharide solution; and

[0144] g. Further passing the purified capsular polysaccharide solution obtained after step (f) through an enhanced cellulose membrane with quaternary ammonium as a ligand to remove process impurities, obtaining a super-purified capsular polysaccharide solution.

[0145] In one embodiment, the present invention provides a method for purifying capsular polysaccharide from Streptococcus pneumoniae serotypes 1 and 5, wherein the method comprises the following steps:

[0146] a. Cultivate bacterial cells under standard growth conditions to obtain a fermentation broth;

[0147] b. Lyse the bacterial cells in the fermentation broth obtained in step (a) to obtain a bacterial cell lysate containing capsular polysaccharide, protein, nucleic acid, cell wall components and other impurities;

[0148] c. Subject the lysate to a preliminary purification process including centrifugation, depth filtration, and diafiltration to obtain a preliminarily treated crude polysaccharide solution;

[0149] d. Control the size of the preliminarily treated crude polysaccharide solution in step (c) using a homogenizer to obtain a size-controlled crude capsular polysaccharide solution;

[0150] e. Pass the size-controlled crude capsular polysaccharide solution obtained in step (d) through an enhanced cellulose membrane having quaternary ammonium as a ligand; then

[0151] f. Contact the solution obtained after passing through the enhanced cellulose membrane having quaternary ammonium as a ligand in step (e) with SiO2 to obtain a purified capsular polysaccharide solution; and

[0152] g. Further pass the purified capsular polysaccharide solution obtained after step (f) through an enhanced cellulose membrane having quaternary ammonium as a ligand to remove process impurities and obtain an ultra-purified capsular polysaccharide solution.

[0153] In yet another embodiment, the purified capsular polysaccharide solution obtained after contact with SiO2 or the filtered polysaccharide solution obtained after treating the purified polysaccharide solution in contact with SiO2 with a depth filter, a carbon filter and a 0.45-micron filter can be further passed through an enhanced cellulose membrane with a quaternary ammonium ligand to remove process impurities for purification.

[0154] In one embodiment, the purified capsular polysaccharide solution can be concentrated and diafiltered using normal saline and water for injection to remove low molecular weight polysaccharides and residual reagents used in the purification process.

[0155] In a preferred embodiment, the purified capsular polysaccharide solution can be concentrated and diafiltered using a 100 kDa molecular weight cut-off membrane.

[0156] The purified and ultrafiltered capsular polysaccharide can be further subjected to membrane filtration to remove bioburden. Generally, the capsular polysaccharide solution is filtered through a 0.45-micron filter and then through a 0.22-micron filter to remove bioburden. However, any suitable membrane can be used for filtration to remove bioburden. The filtered capsular polysaccharide solution can be refrigerated after sampling. Preferably, the capsular polysaccharide solution can be stored at -20 °C after sampling.

[0157] In another embodiment of the present invention, the protein content in the purified capsular polysaccharide is less than 2% of the dry weight of the polysaccharide, the nucleic acid content is less than 2% of the dry weight of the polysaccharide; the endotoxin content is less than 0.5 IU / μg of the dry weight of the polysaccharide.

[0158] In a preferred embodiment of the present invention, the protein content in the purified capsular polysaccharide is less than 1% of the dry weight of the polysaccharide, the nucleic acid content is less than 1% of the dry weight of the polysaccharide; the endotoxin content is less than 0.5 IU / μg of the dry weight of the polysaccharide.

[0159] The product is characterized by sup.1H-NMR data. By determining the protons of the polysaccharide molecules corresponding to the signals, it shows that the product is consistent with the chemical structure. The 1H-NMR spectrum shows a series of well-resolved signals (protons from methyl groups) for quantifying the functional groups in the polysaccharide. The molecular size distribution of the polysaccharide is analyzed by multi-angle laser light scattering (MALLS) technology. The purified polysaccharide complies with WHO specifications.

[0160] In one embodiment, the capsular polysaccharide of the present invention can optionally be further size-controlled. The method of the present invention shows an additional advantage that by minimizing intermolecular and intramolecular associations, it helps prevent the formation of high molecular weight polysaccharide aggregates, thus enabling size control of the polysaccharide at a lower total pressure.

[0161] In one embodiment, the purified capsular polysaccharide of the present invention is used as an immunogen for immunization, with or without further modification. For the purpose of immunization, the capsular polysaccharide is preferably conjugated to a carrier molecule (such as a protein).

[0162] In one embodiment, the carrier protein is selected from the group consisting of tetanus toxoid (TT), diphtheria toxoid (DT), CRM197, Haemophilus influenzae protein D, PhtX, PhtD, PhtDE fusion, detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, Clostridium difficile toxin A or B, and PsaA.

[0163] In a preferred embodiment, the present invention provides an immunogenic composition comprising the purified capsular polysaccharide prepared according to the present invention, which is conjugated to a carrier protein selected from CRM197, PsaA, or PspA, respectively.

[0164] In another preferred embodiment, the present invention provides an immunogenic composition comprising purified capsular polysaccharides from one or more of the following serotypes conjugated to the CRM197 carrier protein: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F.

[0165] In one embodiment, the immunogenic composition comprises conjugated Neisseria meningitidis serogroup Y capsular saccharide (MenY) and / or conjugated Neisseria meningitidis serogroup C capsular saccharide (MenC).

[0166] In another important embodiment of the present invention, the immunogenic composition comprises purified capsular polysaccharides in pure form conjugated to a carrier protein selected from diphtheria toxoid, tetanus toxoid, CRM 197, PsaA, and PspA.

[0167] In some embodiments, the present disclosure provides a Streptococcus pneumoniae polysaccharide-protein conjugate vaccine composition comprising Streptococcus pneumoniae polysaccharides having a molecular weight range of about 100 kDa to about 400 kDa, about 125 kDa to about 425 kDa, about 150 kDa to about 450 kDa, about 175 kDa to about 475 kDa, about 200 kDa to about 500 kDa, about 250 kDa to about 550 kDa, about 300 kDa to about 600 kDa, about 100 kDa to about 1000 kDa, about 200 kDa to about 800 kDa, about 250 kDa to about 600 kDa, about 300 kDa to about 400 kDa, about 70 kDa to about 150 kDa, or about 75 kDa to about 125 kDa.

[0168] The following examples specifically describe the ways of implementing the present invention. However, the embodiments disclosed herein do not limit the scope of the present invention in any way.

[0169] Example

[0170] The present invention will be further illustrated by the following examples, which do not limit the scope of the present invention described above in any way.

[0171] The microorganisms used in the research of the present invention are from the American Type Culture Collection (ATCC), address: 12301 Parklawn Dr., Rockville, Md., USA 20852. The ATCC lists all the serotypes of the present invention and is freely available.

[0172] Example 1: Purifying crude capsular polysaccharide using only SiO2

[0173] This process involves ultrafiltrating the crude polysaccharide obtained from bacterial cell lysis using a 100 kDa molecular weight cut-off (MWCO) membrane at a flow rate of 2 to 5 L / min. The size of the polysaccharide is controlled using a homogenizer. The polysaccharide from serotype 1 is further treated with CTAB, where CTAB is added to the polysaccharide solution at a ratio of 0.2% to 5%. After CTAB treatment, the precipitate is separated by centrifugation and then contacted with Aeroperl. For other serotypes, the size-controlled polysaccharide solution containing impurities is placed in an Aeroperl solution (5.0 g / L to 7.0 g / L) at an incubation temperature of 20 ± 5 °C and treated for no less than 90 minutes. Further, Aeroperl is removed by centrifugation at 12,000 to 14,000 g, and the polysaccharide is collected in the supernatant. The particle size of Aeroperl ranges from 20 μm to 60 μm.

[0174] Table 1: Nucleic acid impurity levels before and after purification of capsular polysaccharide with Aeroperl.

[0175]

[0176] From the data in Table 1, it can be inferred that treatment with Aeroperl alone cannot effectively remove impurities below the permitted regulatory levels.

[0177] Example 2: Purifying crude capsular polysaccharide using an enhanced cellulose membrane with a quaternary ammonium ligand

[0178] This process involves ultrafiltrating the crude capsular polysaccharide with impurities using a membrane with a 100 kDa molecular weight cut-off at a flow rate of 2 to 5 L / min. The size of the polysaccharide is controlled using a homogenizer. The size-controlled polysaccharide is passed through an enhanced cellulose membrane with quaternary ammonium ligands, and collected in the column effluent.

[0179] Table 2: Nucleic acid impurity levels before and after purification of capsular polysaccharide with an enhanced cellulose membrane with quaternary ammonium ligands.

[0180]

[0181] From the data in Table 2, it can be inferred that treatment with an enhanced cellulose membrane with quaternary ammonium ligands alone cannot effectively remove impurities below the permitted regulatory levels.

[0182] Example 3: Purifying crude capsular polysaccharide using an enhanced cellulose membrane with a quaternary ammonium ligand and further exposing to / contacting SiO2

[0183] Inactivate the fermentation broth of Streptococcus pneumoniae serotypes 1 and 5 by adding 0.1% to 0.5% sodium deoxycholate (DOC). Centrifuge the inactivated fermentation broth at a force of 8000 to 10000 g and a flow rate of 200 LPH to 600 LPH. The supernatant is subjected to depth filtration, concentration, and diafiltration against phosphate buffer using a 100 kDa molecular weight cut-off (MWCO) cassette to remove cell debris. Adjust the pH of the clarified supernatant to 5 to 6.

[0184] Filter the concentrated crude capsular polysaccharide through a 0.45-micron filter and control the size using a high-pressure homogenizer. Homogenize the concentrated crude capsular polysaccharide solution within a pressure range of 500 bar to 1000 bar until the polysaccharide particle size reaches 400 kDa to 700 kDa. Further, adjust the pH value of the crude capsular polysaccharide solution to 6.5 to 6.8 and adjust the conductivity of the crude capsular polysaccharide solution to 3 ± 0.2 mS / cm.

[0185] First, wash the enhanced cellulose membrane with a quaternary ammonium as a ligand with 0.5N to 1N NaOH, and then equilibrate it with phosphate-buffered saline (PBS) until the pH reaches 6.5 to 7.8 and the conductivity reaches 3.2 ± 0.3 mS / cm. Use a PBS solution containing 10 mM phosphate and 15 mM NaCl to equilibrate the membrane. Pass the size-controlled crude capsular polysaccharide through the enhanced cellulose membrane with a quaternary ammonium ligand at a flow rate of 2 L / min to 5 L / min. Then, adsorb the effluent solution obtained after passing the size-controlled crude capsular polysaccharide solution through the enhanced cellulose membrane with a quaternary ammonium ligand with Aeroperl at room temperature for 1 to 2 hours. The particle size of Aeroperl is in the range of 20 μm to 60 μm, and the concentration is in the range of 5% to 7%.

[0186] Subsequently, separate Aeroperl from the polysaccharide solution by centrifugation at 12000 - 14000 g and a filtration process using a depth filter, a carbon filter, and a 0.45-micron filter. Optionally, further pass the treated solution after Aeroperl treatment through the enhanced cellulose membrane with a quaternary ammonium ligand.

[0187] Treat again through the enhanced cellulose membrane with a quaternary ammonium as a ligand to further remove process impurities from the filtered polysaccharide solution. All process impurities are selectively bound to the membrane. Collect the polysaccharide solution as the effluent.

[0188] Concentrate and diafilter the purified polysaccharide using a 100 kD molecular weight cut-off membrane with normal saline and water for injection to remove low molecular weight polysaccharides and residual reagents used in the purification process. Filter the concentrated polysaccharide solution through a 0.45-micron filter and then through a 0.22-micron filter to remove bioburden. Store the filtered polysaccharide solution at -20°C.

[0189] The product was characterized using sup.1H-NMR data. By determining the protons of the polysaccharide molecules corresponding to the signals, it was shown that the product was in accordance with the chemical structure. The 1H-NMR spectrum showed a series of well-resolved signals (protons from methyl groups) for quantifying the functional groups in the polysaccharide. The molecular size distribution of the polysaccharide was analyzed using multi-angle laser light scattering (MALLS) technique. The purified capsular polysaccharide complied with WHO specifications.

[0190] Table 3: Impurity levels before and after treating capsular polysaccharide from Streptococcus pneumoniae serotype 1 according to the method of the present invention

[0191]

[0192]

[0193] Table 4: Impurity levels before and after treating capsular polysaccharide from Streptococcus pneumoniae serotype 5 according to the method of the present invention

[0194]

[0195] From the data in Table 3 and Table 4, it can be inferred that treating capsular polysaccharides of Streptococcus pneumoniae serotype 1 and serotype 5 with an enhanced cellulose membrane having a quaternary ammonium ligand and further exposing to SiO2 can effectively remove impurities, making their contents far lower than the permitted regulatory levels, thus obtaining high-quality products.

[0196] Advantage

[0197] The present invention discloses a simple and cost-effective method for purifying capsular polysaccharides without using complex and expensive chromatographic methods and toxic reagents such as phenol.

[0198] Furthermore, the method of the present invention is robust on a commercial scale. The method requires low-cost materials and does not require specialized facilities or handling of hazardous materials.

[0199] In addition, the method of the present invention helps prevent the formation of high-molecular-weight polysaccharide aggregates by minimizing intermolecular and intramolecular associations, thus enabling size control of the polysaccharide at a lower total pressure. It is difficult to control the size of high-molecular-weight polysaccharide aggregates even at a high pressure of about 2000 bar. With the method of the present invention, the size of the capsular polysaccharide can be controlled at a much lower pressure. For example, the size of the capsular polysaccharide can be controlled at about 1200 bar or even lower pressure.

Claims

1. A method for purifying capsular polysaccharide, wherein the method comprises the following steps: a. Culturing bacterial cells under standard growth conditions to obtain a fermentation broth; b. Lysing the bacterial cells in the fermentation broth obtained in step (a) to obtain a bacterial cell lysate containing capsular polysaccharide, protein, nucleic acid, cell wall components and other impurities; c. Controlling the size of the bacterial cell lysate in step (b) using a homogenizer to obtain a size-controlled crude capsular polysaccharide solution; d. Passing the size-controlled crude capsular polysaccharide obtained in step (c) through an enhanced cellulose membrane having quaternary ammonium as a ligand; and e. Contacting the solution obtained after passing through the enhanced cellulose membrane having quaternary ammonium in step (d) with silica (SiO2) and separating the capsular polysaccharide in pure form.

2. The method according to claim 1, wherein the bacteria are selected from, but not limited to, Streptococcus, Staphylococcus, Enterococci, Bacillus, Corynebacterium, Listeria, Erysipelothrix, Clostridium, Haemophilus, Neisseria or Klebsiella.

3. The method according to claim 2, wherein the bacteria are selected from, but not limited to, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Group C&G Streptococcii, Staphylococcus aureus, Haemophilus influenzae, Neisseria meningitidis or Klebsiella pneumoniae.

4. The method according to claim 1, wherein the capsular polysaccharide is isolated from, but not limited to, Streptococcus pneumoniae serotypes selected from the group consisting of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10A, 10B, 10C, 10D, 10F, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F, 16A, 16F, 17A, 17F, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32A, 32F, 33A, 33C, 33D, 33E, 33F, 33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, and 48.

5. The method according to claim 6, wherein the capsular polysaccharide is isolated from Streptococcus pneumoniae serotypes selected from serotypes 1 and 5.

6. The method according to claim 1, wherein the purified capsular polysaccharide solution in step (e) is further treated with activated carbon to remove pigment impurities.

7. The method according to claim 1, wherein the purified capsular polysaccharide in step (e) is further passed through an enhanced cellulose membrane having quaternary ammonium as a ligand to remove process impurities.

8. The method according to claim 1, wherein the purification method is an alcohol-free process and does not use CTAB or other irritating chemicals.

9. The method according to claim 1, wherein the enhanced cellulose membrane having a quaternary ammonium ligand is equilibrated with a buffer until the pH reaches 6.5 to 7.5 and the conductivity reaches 2 to 4 mS / cm.

10. The method according to claim 1, wherein before passing the size-controlled crude capsular polysaccharide solution through the enhanced cellulose membrane having a quaternary ammonium ligand, the pH of the solution is adjusted to the range of 6 to 10, and the conductivity of the solution is adjusted to the range of 1.0 mS / cm to 5.0 mS / cm.

11. The method according to claim 1, wherein the size-controlled crude capsular polysaccharide solution from step (c) passes through the enhanced cellulose membrane at a flow rate of 2 L / min to 10 L / min, preferably 2 L / min to 5 L / min.

12. The method according to claim 1, wherein the particle size of the SiO2 used ranges from 0.01 μm to 200 μm.

13. The method according to claim 12, wherein the particle size of the SiO2 preferably ranges from 20 μm to 60 μm.

14. The method according to claim 1, wherein the amount of SiO2 used ranges from 0.5% (w / v) to 20% (w / v).

15. The method according to claim 1, wherein the amount of SiO2 preferably ranges from 5% (w / v) to 7% (w / v).

16. The method according to claim 1, wherein the contact with SiO2 in step (e) is carried out at a temperature of 15 °C to 60 °C for 10 minutes to 16 hours.

17. The method according to claim 1, wherein SiO2 is removed by centrifugation with a force of 12000 to 14000 g.

18. The method according to claim 1, wherein a lysis agent such as an anionic or cationic detergent is used to lyse the fermentation broth.

19. The method according to claim 1, wherein the lysis agent is selected from, but not limited to, sodium deoxycholate (DOC), N-lauroylsarcosine (NLS), sodium chenodeoxycholate, and saponins.

20. An immunogenic composition comprising the purified capsular polysaccharide prepared according to claim 1, wherein the capsular polysaccharide is conjugated to a carrier protein selected from diphtheria toxoid, tetanus toxoid, CRM 197, PsaA, and PspA.

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