Purification of capsular polysaccharides

Abstract

The present invention relates generally to an improved method for purifying capsular polysaccharides. In particular, 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 bacteria. More specifically, the present invention involves passing a crude capsular polysaccharide solution through a reinforced cellulose membrane containing a quaternary ammonium salt, and then contacting the solution with silicon dioxide (SiO2) to isolate the capsular polysaccharide in a pure form. The purified capsular polysaccharide is useful in the preparation of vaccines containing the polysaccharide alone or conjugated to a protein.

Description

[Technical Field]

[0001] The present invention relates generally to an improved method for purifying capsular polysaccharides. In particular, 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 bacteria. More specifically, the present invention involves passing a crude capsular polysaccharide solution through a reinforced cellulose membrane containing a quaternary ammonium salt, and then contacting the solution with silicon dioxide (SiO2) to isolate the capsular polysaccharide in pure form. The purified capsular polysaccharide is useful in the preparation of vaccines containing the polysaccharide alone or conjugated to a protein. [Background technology]

[0002] The manufacturing process of a vaccine is crucial at every stage to ensure its safety for human use. Polysaccharides are carbohydrates used in several industrial applications, including as thickeners, gelling agents, emulsifiers, and delivery systems in numerous commercial products. Capsular polysaccharides present on bacterial cells can be used as components of vaccinations. Vaccination with purified capsular polysaccharide formulations prevents diseases caused by microorganisms such as Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae type b, and Salmonella typhi by inducing the respective immune responses. This makes them an important component in vaccine design.

[0003] Capsular polysaccharides have also proven useful in eliciting immune responses, especially when conjugated to carrier proteins. Conjugate vaccines provide improved immunogenic responses in children, immunocompromised individuals, and the elderly. Polysaccharides conjugated to proteins such as CRM197, tetanus toxoid, diphtheria toxoid, and other surface proteins have been shown to be highly immunogenic.

[0004] All such immunogenic or vaccine preparations approved for human use require capsular polysaccharides in a highly purified form. Capsular polysaccharides are present on the outer surface of bacterial cells. During separation of capsular polysaccharides from cells, cellular components such as nucleic acids, proteins, and cell walls are released. Biosynthetic products produced during fermentation as well as medium nutrients contribute as contaminants. The capsular polysaccharide isolation / purification process requires multiple steps, ranging from chromatography to filtration, detergent treatment, solvent treatment, and enzymatic treatment to hydrolyze nucleic acids, proteins, polysaccharides, etc.

[0005] The following references disclose various methods for removing proteins and other impurities from capsular polysaccharides: Canadian Patent No. 1206905 describes a method using toxic organic solvents such as phenol, butanol, toluene and chloroform, and using the detergent Cetavlon (CTAB).

[0006] EP 0497525 discloses a method for hydrolyzing samples using thermal hydrolysis and sodium acetate. US Patent No. 5,847,112 describes a method for precipitation of polysaccharides using multiple isopropyl alcohol precipitations and Cetavlon.

[0007] US Patent Publication No. 20060228380 discloses a method involving Cetavlon for precipitation of polysaccharides, carbon filters for removal of nucleic acids, and potassium iodide for precipitation of Cetavlon.

[0008] WO 2006 / 082527 A2 discloses a method for purifying the capsular polysaccharide of group B Streptococcus (agalactiae) in which the saccharide is first treated with an aqueous mixture of alcohol and calcium salt, followed by precipitation with a cationic detergent. Indian Patent Application No. 1572 / MUM / 2010 discloses a purification method for removing protein contaminants, which involves treating a nuclease-treated polysaccharide solution with a mixture of detergent and saline, followed by centrifugation, diafiltration and chromatography.

[0009] International Patent Publication No. 2012 / 127485 A1 discloses an alcohol- and CTAB-free method for purifying pneumococcal polysaccharides, which relies on chromatographic separation of capsular polysaccharides (PnPs) based on differences in net surface charge.

[0010] Referring to the prior art, it can be seen that removing process impurities from capsular polysaccharides is very difficult as both are negatively charged. The methods disclosed in the above prior art involve many complicated steps, which adversely affect the yield, quality, stability, processing time and process operation. Furthermore, these methods have high operating costs, require high skill to perform and require a considerable amount of time to reduce the impurity levels.

[0011] Furthermore, the methods disclosed in the prior art are inefficient in removing process impurities from bacterial capsular polysaccharides when the initial input is high or when the impurities are produced in aggregated form. Therefore, there is a need for an improved method for removing impurities from complex cell lysates. Through tireless efforts, the inventors have discovered that impurities can be effectively removed by using a reinforced cellulose membrane with quaternary ammonium ligands in conjunction with silicon dioxide. An optimal pH is used to bind strongly negatively charged DNA, endotoxins, and host cell proteins to the reinforced cellulose membrane with quaternary ammonium ligands.

[0012] The method we have developed is simple, efficient, and easily scalable. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] Canadian Patent No. 1206905 [Patent Document 2] European Patent No. 0497525 [Patent Document 3] U.S. Patent No. 5,847,112 [Patent Document 4] U.S. Patent Publication No. 20060228380 [Patent Document 5] International Patent Publication No. 2006 / 082527 A2 [Patent Document 6] Indian Patent Application No. 1572 / MUM / 2010 [Patent Document 7] International Patent Publication No. 2012 / 127485 Al Summary of the Invention [Problem to be solved by the invention]

[0014] 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 bacteria, and to provide purified capsular polysaccharides that meet compendial requirements.

[0015] It is yet another object of the present invention to provide a simple, cost-effective method for purifying capsular polysaccharides without the use of complex and expensive chromatographic methods or toxic reagents such as phenol.

[0016] Furthermore, another important object of the present invention is to provide a method for purifying capsular polysaccharides on a commercial scale in a robust manner, requiring only inexpensive process materials, and without specialized equipment or the disposal of hazardous substances. [Means for solving the problem]

[0017] The present invention provides a method for purifying capsular polysaccharides, said method comprising the steps of: 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 polysaccharides, proteins, nucleic acids, cell wall components and other impurities; c. subjecting the bacterial cell lysate obtained from step (b) to sizing using a homogenizer to obtain a sized crude capsular polysaccharide solution; d. Passing the sized crude capsular polysaccharide obtained in step (c) through a reinforced cellulose membrane having a quaternary ammonium ligand; and e. Contacting the solution obtained in step (d) after passing through a reinforced cellulose membrane having a quaternary ammonium salt with SiO2 to isolate the capsular polysaccharide in pure form.

[0018] In one embodiment, the method of the present invention does not require complex and expensive chromatographic procedures or toxic reagents such as phenol for the purification of capsular polysaccharides. In another embodiment of the present invention, the capsular polysaccharide is isolated from a Streptococcus pneumoniae serotype selected from serotypes 1 and 5.

[0019] In yet another embodiment, the purification method of the present invention is an alcohol-free method and does not use CTAB or other harsh chemicals. DETAILED DESCRIPTION OF THE INVENTION

[0020] The embodiments described herein can be more readily understood by reference to the following detailed description and examples. The elements and methods described herein are merely illustrative of the principles of the invention and are not limited to the specific embodiments shown in the detailed description and examples. Numerous modifications and adaptations will be readily apparent to those skilled in the art without departing from the spirit and scope of the invention.

[0021] 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 the method belongs. Although any methods, devices, kits, reagents, or compositions similar or equivalent to those described herein can also be used in the practice or testing of the methods, representative and illustrative methods and compositions are described herein. It should be understood that certain features of methods that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of methods and compositions that are, for brevity, described in the context of a single embodiment may be provided separately or in any suitable subcombination.

[0022] It is noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include their plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement shall serve as an antecedent basis for the use of exclusive terms such as "solely," "only," and the like in connection with the recitation of claim elements or the use of a "negative" limitation. As used herein, the term "sized" or "sizing" refers to reducing the size of natural polysaccharides by various methods.

[0023] A "lysing agent" is any agent that aids in the breakdown of cell walls, such as a detergent. As used herein, the term "detergent" refers to any anionic or cationic detergent that is capable of inducing the lysis of bacterial cells.

[0024] The terms "exposing" or "contacting" refer to the incubation of a capsular polysaccharide preparation with other components to treat the sample and remove impurities in order to produce pure polysaccharide.

[0025] Those skilled in the art will appreciate, upon reading this disclosure, that each of the individual embodiments described and illustrated herein has separate components and features that may be readily separated from or combined with the features of any other embodiment without departing from the scope or spirit of the method. Any described method can be carried out in the order of events described or in any other order that is logically possible.

[0026] The practice of the present invention will employ, unless otherwise indicated, conventional methods of virology, immunology, microbiology and molecular biology within the skill of the art, which techniques are fully explained in the available literature.

[0027] Removal of process impurities from capsular polysaccharides is a challenging process because both the impurities and the capsular polysaccharides are negatively charged. The pH value at which a biomolecule has no net charge is its isoelectric point. Therefore, in the present invention, an optimal pH range is used to bind strongly negatively charged DNA, endotoxins, and host cell proteins to reinforced cellulose membranes with quaternary ammonium ligands.

[0028] The present invention provides an improved method for purifying capsular polysaccharides, the source of which is Gram-negative and Gram-positive bacteria. The improved method involves passing a crude capsular polysaccharide solution through a reinforced cellulose membrane containing a quaternary ammonium salt and contacting the solution with SiO. After passing through the reinforced cellulose membrane containing the quaternary ammonium salt and exposure to SiO, the resulting solution is enriched in polysaccharides and reduced in one or more impurities, such as proteins, nucleic acids, cell wall polysaccharides, and other cellular materials.

[0029] The capsular polysaccharides obtained according to the invention are in (substantially) pure form. An important aspect of the present invention relates to the removal of impurities from complex bacterial cell lysates containing crude capsular polysaccharides. It involves the use of silicon dioxide in conjunction with a reinforced cellulose membrane bearing quaternary ammonium ligands, which results in effective removal of impurities. Importantly, a significant reduction in process impurities was observed without loss of capsular polysaccharides.

[0030] In another aspect of the present invention, the purified capsular polysaccharide met all release testing specifications as per the World Health Organization Technical Report Series 927. Additionally, the WHO specifications for the purified capsular polysaccharide are as follows:

[0031] Protein content Dependent on serotype polysaccharide, but generally less than 2% of the dry weight of polysaccharide. Levels are less than 2% for serotype 1 and less than 3% for serotype 5.

[0032] Nucleic acid content : Not more than 2% of the dry weight of polysaccharides. Endotoxin content : Less than 0.5 IU / μg polysaccharide (per μg polysaccharide).

[0033] Some important aspects of the present invention are as follows: An important aspect of the present invention relates to a method for purifying crude capsular polysaccharides, said method comprising the steps of:

[0034] i. passing a solution of bacterial cell lysate containing capsular polysaccharides, proteins, nucleic acids, cell wall components and other impurities through a reinforced cellulose membrane having a quaternary ammonium ligand; and ii. Contacting the solution obtained in step (i) after passing through a reinforced cellulose membrane having a quaternary ammonium salt with SiO2 to isolate the capsular polysaccharide in pure form.

[0035] A preferred embodiment of the present invention relates to a method for purifying crude capsular polysaccharides, said method comprising the steps of: 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 polysaccharides, proteins, nucleic acids, cell wall components and other impurities; c. subjecting the bacterial cell lysate obtained from step (b) to sizing using a homogenizer to obtain a sized crude capsular polysaccharide solution; d. Passing the sized crude capsular polysaccharide solution obtained in step (c) through a reinforced cellulose membrane having a quaternary ammonium group as a ligand; and e. Contacting the solution obtained in step (d) after passing through a reinforced cellulose membrane having a quaternary ammonium salt with SiO2 to isolate the capsular polysaccharide in pure form.

[0036] In one embodiment, the capsular polysaccharides of the present invention are isolated from bacteria. In one embodiment of the present invention, the bacterium is a Gram-positive bacterium. In one aspect, the bacterium is selected from, but is not limited to, Streptococcus, Staphylococcus, Enterococci, Bacillus, Corynebacterium, Listeria, Erysipelothrix, or Clostridium. In a further aspect, the bacterium is selected from, but is not limited to, Streptococcus pneumoniae, Group A Streptococcus pyogenes, Group B Streptococcus agalactiae, Group C & G Streptococcus, or Staphylococcus aureus.

[0037] In another embodiment of the present invention, the bacterium is a Gram-negative bacterium. In one aspect, the bacterium is selected from, but is not limited to, the genera Haemophilus, Neisseria, or Klebsiella. In another aspect, the bacterium is selected from, but is not limited to, Haemophilus influenzae, Neisseria meningitidis, or Klebsiella pneumoniae.

[0038] In another important aspect of the present invention, the bacterium is selected from, but is not limited to, the genera Streptococcus, Staphylococcus, Enterococcus, Bacillus, Corynebacterium, Listeria, Erysipelothricus, Clostridium, Haemophilus, Neisseria, or Klebsiella.

[0039] In yet another embodiment of the present invention, the bacterium is selected from, but is not limited to, Streptococcus pneumoniae, Group A Streptococcus pyogenes, Group B Streptococcus agalactiae, Group C and G Streptococcus, Staphylococcus aureus, Haemophilus influenzae, Neisseria meningitidis, or Klebsiella pneumoniae.

[0040] In another embodiment of the invention, the capsular polysaccharide is selected from the group consisting of 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, 20A, 20B, 21A, 21B, 21C, 21D, 22A, 22B, 22F, 23A, 23B, 24A, 24C, 24F, 25A, 25C, 25B, 26A, 26F, 27A, 27F, 28A, 28B, 29A, 29B, 29C, 29F, 30A, 30B, 31A, 31B, 31C, 31D, 31E, 32A, 32B, 32F, 33A, 33C, 34A, 34B, 35A, 35C, 35B, 35F, 36A, 36F, 37A, 37F, and is isolated from a Streptococcus pneumoniae serotype selected from the group including, but not limited to, 0, 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.

[0041] In an exemplary embodiment of the invention, the capsular polysaccharide is isolated from a Streptococcus pneumoniae serotype selected from serotypes 1 and 5. In another embodiment of the present invention, the improved purification method is an alcohol-free method and does not use CTAB or other harsh chemicals.

[0042] In one embodiment of the invention, the capsular polysaccharide is produced by growing the bacteria in a medium (eg, a solid medium or, preferably, a liquid medium). In another embodiment of the invention, the starting material for the process of the invention is a bacterial culture, preferably a liquid bacterial culture (e.g., a fermentation broth).

[0043] In an exemplary embodiment, bacterial cultures are grown in liquid MH medium. The bacterial cultures are grown in fermentors under controlled parameters, including a temperature range of 35±2°C, a pH range of 7.1±0.3, and a backpressure of 0.2-1.0 bar. Optical density is monitored every 30-60 minutes. Fermentation is terminated when the culture enters stationary or decay phase. The bacterial cells are then processed using a variety of methods, including, but not limited to, cell lysis, centrifugation, depth filtration, concentration / diafiltration procedures, and precipitation processes, to produce capsular polysaccharide.

[0044] 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 continuous centrifugation. The supernatant is subjected to depth filtration, concentration, diafiltration using a phosphate buffer, and optionally ultrafiltration. The solution obtained after pre-treatment of the bacterial cell lysate is called a crude polysaccharide solution.

[0045] In one embodiment, the fermentation broth is lysed using a lysing agent. In a preferred embodiment of the present invention, the lysing agent is selected from, but not limited to, sodium deoxycholate (DOC), N-lauryl sarcosine (NLS), sodium chenodeoxycholate, and saponin.

[0046] In another embodiment of the present invention, the fermentation broth is treated with a 0.1-0.5% w / v DOC solution to lyse the cells in the fermentation broth. In one embodiment, the capsular polysaccharides in the crude capsular polysaccharide solution or bacterial cell lysate are sized before passing the solution through a reinforced cellulose membrane having a quaternary ammonium group as a ligand. The solution obtained after subjecting the crude capsular polysaccharide or bacterial cell lysate to sizing is referred to as the post-sized crude polysaccharide solution.

[0047] In another embodiment, to avoid cross-linking of process impurities with product-derived impurities and to make the capsular polysaccharide resistant to sizing, high molecular weight product aggregates in bacterial cell lysates containing capsular polysaccharides are removed by introducing a sizing step using chemical sizing methods or treatment with a high-pressure homogenizer before proceeding to the purification process.

[0048] In an exemplary embodiment, the capsular polysaccharide is microfluidized, Emulsiflex TM Size reduction is accomplished by various mechanical means known in the art, such as high-pressure homogenization, sonication, or high-pressure techniques such as Gaulin homogenization. Homogenization involves pumping the process stream through a channel of sufficiently small dimensions to achieve high shear rates. Shear rates are increased by using greater applied homogenization pressures, and exposure times are increased by recirculating the feed stream through the homogenizer.

[0049] In one embodiment, homogenization is carried out at a pressure of at least 500 bar until polysaccharides of the desired size are obtained. In one embodiment of the present invention, homogenization is preferably carried out at 500 to 1000 Bar and a flow rate of 50 to 200 L / hour.

[0050] In one embodiment, the molecular weight of the polysaccharide after sizing ranges from 50 kDa to 1000 kDa. In another embodiment, the molecular weight of the polysaccharide after sizing ranges from about 400 kDa to 700 kDa.

[0051] In a preferred embodiment, the molecular weight of the polysaccharide after sizing ranges from about 150 kDa to 350 kDa. In one embodiment, the crude capsular polysaccharide solution after sizing is passed through a reinforced cellulose membrane bearing quaternary ammonium ligands.

[0052] In one embodiment, the reinforced cellulose membrane with quaternary ammonium as a ligand is used in any form such as a matrix or filter capsule. In one embodiment, the reinforced cellulose membrane bearing quaternary ammonium ligands is first washed with alkali and then equilibrated with a buffer solution before use.

[0053] In one embodiment, the membrane is first sanitized with 1N NaOH for 30 minutes or more. In another embodiment of the invention, sanitization of a reinforced cellulose membrane bearing quaternary ammonium ligands is carried out with 0.5 M NaOH, followed by equilibration with phosphate buffered saline.

[0054] In another embodiment of the invention, a reinforced cellulose membrane with quaternary ammonium ligands is equilibrated with a buffer solution until the pH reaches 6.5-7.5 and the conductivity reaches 2-4 mS / cm.

[0055] In yet another embodiment, a reinforced cellulose membrane with quaternary ammonium ligands is equilibrated with a buffer solution until the pH reaches 6.5-6.8 and the conductivity reaches 3.2±0.3 mS / cm.

[0056] In one embodiment, the pH of the sizing crude capsular polysaccharide solution is adjusted to a range of about 6.0 to 10.0 before passing the solution through a reinforced cellulose membrane bearing quaternary ammonium ligands.

[0057] In a preferred embodiment, the pH of the sized crude capsular polysaccharide solution is adjusted to a range of about 6.5 to 6.8 before passing the solution through a reinforced cellulose membrane bearing quaternary ammonium ligands.

[0058] In one embodiment of the invention, the conductivity of the sized crude capsular polysaccharide solution is adjusted to a range of about 1.0 mS / cm to 5.0 mS / cm before passing the solution through a reinforced cellulose membrane bearing quaternary ammonium ligands.

[0059] In a preferred embodiment of the present invention, the conductivity of the crude capsular polysaccharide solution after sizing is adjusted to a range of about 2.8 to 3.2 mS / cm before passing the solution through a reinforced cellulose membrane bearing quaternary ammonium ligands.

[0060] In one embodiment of the present invention, the conductivity of the crude capsular polysaccharide solution after sizing is adjusted using a 0.5-5 M NaCl or 0.5-5 M KCl solution. In one embodiment of the invention, the sized crude capsular polysaccharide solution is passed through the membrane at a flow rate ranging from about 2 L / min to 10 L / min.

[0061] In a preferred embodiment of the present invention, the sized crude capsular polysaccharide solution is passed through the membrane at a flow rate ranging from about 3 L / min to 5 L / min. In another embodiment of the invention, the sized crude capsular polysaccharide solution is passed through a reinforced cellulose membrane at a flow rate of 2-5 L / min.

[0062] In one embodiment of the present invention, the flow-through solution obtained by passing the crude capsular polysaccharide solution after sizing through a reinforced cellulose membrane bearing quaternary ammonium ligands is exposed to or contacted with SiO2.

[0063] In one embodiment, residual SiO2 after contact is removed by centrifugation. In one embodiment of the invention, the crude capsular polysaccharide solution after sizing is passed through a reinforced cellulose membrane bearing quaternary ammonium ligands, and the resulting solution is then exposed to or contacted with SiO2 without any intermediate treatment or purification steps.

[0064] In one embodiment, the SiO2 used is in various forms / particle sizes such as particulates ranging from 0.01 μm to 200 μm. In another embodiment, the SiO2 used is in various forms / particle sizes, such as fine particles, preferably in the range of 3 μm to 40 μm.

[0065] In a preferred embodiment, the particle size of SiO2 is in the range of 10 μm to 80 μm, more preferably in the range of 20 μm to 60 μm. In one embodiment of the present invention, the amount of SiO2 used may range from 0.5 to 20% (w / v).

[0066] In a preferred embodiment, the amount of SiO2 ranges from 5% to 7% (w / v) (50 g / L to 70 g / L). In one embodiment, the SiO2 used may be prepared by heating above about 60°C for at least 1 hour and cooling before use.

[0067] In another embodiment, the SiO2 used may be pyrogenated or depyrogenated. In another embodiment, the pH of the solution obtained by passing the crude capsular polysaccharide solution after sizing through a reinforced cellulose membrane having quaternary ammonium ligands is maintained in the acidic to alkaline range, preferably in the range of 3.0 to 9.0, during contact with SiO2.

[0068] In another embodiment, the pH of the solution obtained by passing the crude capsular polysaccharide solution after sizing through a reinforced cellulose membrane with quaternary ammonium ligands is adjusted using an acid such as acetic acid, phosphoric acid, formic acid, or hydrochloric acid, and an alkali such as sodium hydroxide, potassium hydroxide, or ammonium hydroxide.

[0069] In a preferred embodiment, the pH of the solution obtained by passing the sized crude capsular polysaccharide solution through a reinforced cellulose membrane having quaternary ammonium ligands is maintained in the range of 5.0 to 7.0 during contact with SiO2.

[0070] In one embodiment, the crude capsular polysaccharide solution after sizing is passed through a reinforced cellulose membrane having quaternary ammonium ligands, and the resulting solution is contacted or exposed to SiO2 at a temperature ranging from 15°C to 60°C for 10 minutes to 16 hours.

[0071] In another embodiment, the crude capsular polysaccharide solution after sizing is passed through a reinforced cellulose membrane having quaternary ammonium ligands, and the resulting solution is contacted or exposed to SiO2 at room temperature (22±4°C) for 1 to 2 hours.

[0072] In yet another embodiment, the crude capsular polysaccharide solution after sizing is passed through a reinforced cellulose membrane having quaternary ammonium ligands, and the resulting solution is contacted or exposed to SiO2 at a temperature of 50±5°C for 60 to 80 minutes.

[0073] In a preferred embodiment of the present invention, the contact with SiO2 is preferably carried out at a temperature in the range of 15°C to 25°C for a time in the range of 60 minutes to 80 minutes under stirring. In another embodiment of the present invention, the SiO2 was removed using centrifugation at 12000-14000 g force.

[0074] In another embodiment of the invention, Aeroperl® is used, although a common brand name for commercially available SiO 2 (silicon dioxide) is Aerosil®.

[0075] In another embodiment, the invention includes treating the purified capsular polysaccharide solution with activated carbon to remove colored impurities, which treatment is performed after exposure to SiO2. In one aspect, the present invention provides a method for purifying capsular polysaccharides from serotypes 1 and 5 of Streptococcus pneumoniae, said method comprising the steps of:

[0076] 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 polysaccharides, proteins, nucleic acids, cell wall components and other impurities; c. subjecting the bacterial cell lysate of step (b) to sizing using a homogenizer to obtain a sized crude capsular polysaccharide solution; d. Passing the sized crude capsular polysaccharide solution obtained in step (c) through a reinforced cellulose membrane having a quaternary ammonium group as a ligand; and e. Contacting the solution obtained in step (d) after passing through a reinforced cellulose membrane having quaternary ammonium as a ligand with SiO2 to isolate the capsular polysaccharide in pure form.

[0077] In one aspect, the present invention provides a method for purifying capsular polysaccharides, said method comprising the steps of: 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 polysaccharides, proteins, nucleic acids, cell wall components and other impurities; c. subjecting the lysed broth to pre-purification steps including centrifugation, depth filtration, and diafiltration to obtain a pre-treated crude polysaccharide solution; d. subjecting the pretreated crude polysaccharide solution of step (c) to sizing using a homogenizer to obtain a sized crude capsular polysaccharide solution; e. passing the sized crude capsular polysaccharide solution obtained in step (d) through a reinforced cellulose membrane having a quaternary ammonium group as a ligand; and thereafter f. Contacting the solution obtained in step (e) after passing through a reinforced cellulose membrane having quaternary ammonium as a ligand with SiO2 to isolate the capsular polysaccharide in pure form.

[0078] In one aspect, the present invention provides a method for purifying capsular polysaccharides from serotypes 1 and 5 of Streptococcus pneumoniae, said method comprising the steps of: 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 polysaccharides, proteins, nucleic acids, cell wall components and other impurities; c. subjecting the lysed broth to pre-purification steps including centrifugation, depth filtration, and diafiltration to obtain a pre-treated crude polysaccharide solution; d. subjecting the pretreated crude polysaccharide solution of step (c) to sizing using a homogenizer to obtain a sized crude capsular polysaccharide solution; e. passing the sized crude capsular polysaccharide solution obtained in step (d) through a reinforced cellulose membrane having a quaternary ammonium group as a ligand; and thereafter f. Contacting the solution obtained in step (e) after passing through a reinforced cellulose membrane having quaternary ammonium as a ligand with SiO2 to isolate the capsular polysaccharide in pure form.

[0079] In one embodiment, the diafiltration in step (c) above is carried out against a phosphate buffer using a 100 kDa MWCO cassette. 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.

[0080] In one embodiment, the purified capsular polysaccharide solution can be further passed through a reinforced cellulose membrane with quaternary ammonium as a ligand to remove process impurities and obtain an ultra-purified capsular polysaccharide solution.

[0081] In one aspect, the present invention provides a method for purifying capsular polysaccharides, said method comprising the steps of: 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 polysaccharides, proteins, nucleic acids, cell wall components and other impurities; c. subjecting the lysed broth to pre-purification steps including centrifugation, depth filtration, and diafiltration to obtain a pre-treated crude polysaccharide solution; d. subjecting the pretreated crude polysaccharide solution of step (c) to sizing using a homogenizer to obtain a sized crude capsular polysaccharide solution; e. passing the sized crude capsular polysaccharide solution obtained in step (d) through a reinforced cellulose membrane having a quaternary ammonium group as a ligand; and thereafter f. contacting the solution obtained in step (e) after passing through a reinforced cellulose membrane having a quaternary ammonium group as a ligand with SiO2 to obtain a purified capsular polysaccharide solution; and g. A step of passing the purified capsular polysaccharide solution obtained after step (f) through a reinforced cellulose membrane having a quaternary ammonium group as a ligand to remove process impurities and obtain an ultra-purified capsular polysaccharide solution.

[0082] In one aspect, the present invention provides a method for purifying capsular polysaccharides from serotypes 1 and 5 of Streptococcus pneumoniae, said method comprising the steps of: 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 polysaccharides, proteins, nucleic acids, cell wall components and other impurities; c. subjecting the lysed broth to pre-purification steps including centrifugation, depth filtration, and diafiltration to obtain a pre-treated crude polysaccharide solution; d. subjecting the pretreated crude polysaccharide solution of step (c) to sizing using a homogenizer to obtain a sized crude capsular polysaccharide solution; e. passing the sized crude capsular polysaccharide solution obtained in step (d) through a reinforced cellulose membrane having a quaternary ammonium group as a ligand; and thereafter f. contacting the solution obtained in step (e) after passing through a reinforced cellulose membrane having a quaternary ammonium group as a ligand with SiO2 to obtain a purified capsular polysaccharide solution; and g. A step of passing the purified capsular polysaccharide solution obtained after step (f) through a reinforced cellulose membrane having a quaternary ammonium group as a ligand to remove process impurities and obtain an ultra-purified capsular polysaccharide solution.

[0083] In yet another embodiment, the purified capsular polysaccharide solution obtained after contact with SiO2, or the filtered polysaccharide solution obtained after depth filtering, carbon filtering, and 0.45 micron filtering of the SiO2-contacted purified polysaccharide solution, can be further purified by passing it through a reinforced cellulose membrane bearing quaternary ammonium ligands to remove process impurities.

[0084] In one embodiment, the purified capsular polysaccharide solution can be concentrated and subjected to diafiltration using saline and water for injection to remove low molecular weight polysaccharides and residual chemicals used in the purification process.

[0085] In a preferred embodiment, the purified capsular polysaccharide solution can be subjected to concentration and diafiltration using a 100 kDa molecular weight cutoff membrane. The purified and ultrafiltered capsular polysaccharide can be further subjected to membrane filtration to remove bioburden. Typically, the capsular polysaccharide solution is filtered through a 0.45 micron filter followed by 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 is stored at -20°C after sampling.

[0086] In another embodiment of the invention, the purified form of capsular polysaccharide has a protein content of less than 2% of the dry weight of the polysaccharide, a nucleic acid content of less than 2% of the dry weight of the polysaccharide, and an endotoxin content of less than 0.5 IU / μg of the dry weight of the polysaccharide.

[0087] In a preferred embodiment of the invention, the purified capsular polysaccharide has a protein content of less than 1% of the dry weight of the polysaccharide, a nucleic acid content of less than 1% of the dry weight of the polysaccharide, and an endotoxin content of less than 0.5 IU / μg of the dry weight of the polysaccharide.

[0088] The product was characterized using sup.1H-NMR, and the data showed that the assignment of signals assigned to the protons of the polysaccharide molecule was consistent with the chemical structure. The 1H-NMR spectrum showed a series of well-resolved signals (protons from methyl groups) for quantification of the functional groups of the polysaccharide. Multi-angle light scattering (MALLS) was used to profile the molecular size distribution of the polysaccharide. The purified polysaccharide conformed to WHO standards.

[0089] In one embodiment, the capsular polysaccharides of the present invention can be further subjected to sizing. The method of the present invention also offers the additional advantage of helping to prevent the formation of high molecular weight polysaccharide aggregates by minimizing inter- and intramolecular bonds, thereby allowing for sizing of the polysaccharides at significantly lower overall pressures.

[0090] In one embodiment, the purified capsular polysaccharides of the invention, with or without further modification, are used as immunogens for use in vaccination. For vaccination purposes, the capsular polysaccharides are suitably conjugated to a carrier molecule such as a protein.

[0091] In one embodiment, the carrier protein is selected from the group including tetanus toxoid (TT), diphtheria toxoid (DT), CRM197, H. influenzae protein D, PhtX, PhtD, PhtDE fusions, detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, Clostridium difficile toxin A or B, and PsaA.

[0092] In a preferred embodiment, the present invention provides an immunogenic composition comprising purified capsular polysaccharides produced according to the present invention individually conjugated to a carrier protein selected from CRM197, PsaA or PspA.

[0093] In yet another preferred embodiment, the present invention provides an immunogenic composition comprising purified capsular polysaccharides from one or more of serotypes 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 conjugated to CRM197 carrier protein.

[0094] In one aspect, the immunogenic composition comprises conjugated N. meningitidis serogroup Y capsular saccharide (MenY) and / or conjugated meningococcal serogroup C capsular saccharide (MenC).

[0095] In another important embodiment of the present invention, the immunogenic composition comprises purified capsular polysaccharide in pure form conjugated to a carrier protein selected from diphtheria toxoid, tetanus toxoid, CRM197, PsaA, and PspA.

[0096] In some aspects, the present disclosure provides pneumococcal polysaccharide-protein conjugate vaccine compositions comprising a pneumococcal polysaccharide having a molecular weight in the 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 Da, or about 75 kDa to about 125 kDa.

[0097] The following examples are provided to specifically illustrate the manner in which the present invention may be practiced, however, the embodiments disclosed herein are not intended to limit the scope of the present invention in any way. [Example]

[0098] Without in any way limiting the scope of the invention described above, the invention will be further illustrated through the examples provided below. The microorganisms used for the studies in this invention were obtained from the American Type Culture Collection (ATCC), 12301 Parklawn Dr., Rockville, Md., USA 20852. The ATCC lists all serotypes of this invention as freely available.

[0099] Example 1: SiO 2 Purification of crude capsular polysaccharides by ethanol alone This method involves diafiltration of crude polysaccharides obtained from bacterial cell lysis using a 100 kDa molecular weight cutoff (MWCO) membrane at a flow rate of 2-5 L / min. The polysaccharides are then sized using a homogenizer. Polysaccharides derived from serotype 1 are further treated with CTAB (CTAB is added to the polysaccharide solution at 0.2%-5%). After CTAB treatment, the pellet is separated by centrifugation and then contacted with Aeroperl. For other serotypes, the sized, impure polysaccharide solution is exposed to Aeroperl solution (5.0-7.0 g / L) at an incubation temperature of 20±5°C for 90 minutes or more. The Aeroperl is then removed by centrifugation at 12,000-14,000 g. The polysaccharides are recovered in the supernatant. The particle size of Aeroperl ranges from 20-60 μm.

[0100] Table 1: Nucleic acid impurity levels before and after purification of capsular polysaccharides by Aeroperl

[0101] [Table 1]

[0102] From the data in Table 1, it can be inferred that Aeroperl treatment alone is not effective in removing impurities below the permitted regulatory levels. Example 2: Purification of crude capsular polysaccharides by reinforced cellulose membranes with quaternary ammonium ligands The method involves diafiltration of impure crude capsular polysaccharide using a 100 kDa molecular weight cutoff (MWCO) membrane at a flow rate of 2-5 L / min. The polysaccharide is subjected to sizing using a homogenizer. The sized polysaccharide is passed through a reinforced cellulose membrane bearing quaternary ammonium ligands. The polysaccharide is collected in the column effluent.

[0103] Table 2: Nucleic acid impurity levels before and after purification of capsular polysaccharides through reinforced cellulose membranes with quaternary ammonium ligands.

[0104] [Table 2]

[0105] From the data in Table 2, it can be inferred that treatment with reinforced cellulose membranes bearing quaternary ammonium ligands alone is not effective in removing impurities below the permitted regulatory levels. Example 3: Reinforced cellulose membrane with quaternary ammonium ligands and further SiO 2 Purification of crude capsular polysaccharides by exposure / contact with Fermentation broth of Streptococcus pneumoniae strains from serotypes 1 and 5 is inactivated by the addition of 0.1-0.5% sodium deoxycholate (DOC). The inactivated fermentation broth is centrifuged at 8,000-10,000 g force and a flow rate of 200-600 LPH. The supernatant is concentrated by depth filtration and diafiltered against phosphate buffer using a 100 kDa MWCO cassette to remove cell debris. The pH of the clarified supernatant is adjusted to 5-6.

[0106] The concentrated crude capsular polysaccharide is filtered through a 0.45 micron filter and subjected to sizing using a high-pressure homogenizer. The concentrated and sized crude polysaccharide solution is homogenized at a pressure ranging from 500 to 1000 bar until the polysaccharide size reaches 400 to 700 kDa. The pH of the sized crude capsular polysaccharide solution is then adjusted to a range of 6.5 to 6.8, and the conductivity of the sized crude capsular polysaccharide solution is adjusted to 3±0.2 mS / cm.

[0107] A reinforced cellulose membrane with quaternary ammonium ligands was first washed with 0.5–1 N NaOH and then equilibrated with phosphate-buffered saline (PBS) until the pH reached 6.5–7.8 and the conductivity reached 3.2 ± 0.3 mS / cm. PBS containing 10 mM phosphate in 15 mM NaCl was used for membrane equilibration. The sized crude capsular polysaccharide was passed through the reinforced cellulose membrane with quaternary ammonium ligands at a flow rate of 2–5 L / min. The effluent obtained by passing the sized crude capsular polysaccharide solution through the reinforced cellulose membrane with quaternary ammonium ligands was then subjected to adsorption with Aeroperl at room temperature for 1–2 hours. The particle size of Aeroperl ranged from 20–60 μm, and the concentration ranged from 5–7%.

[0108] The Aeroperl is then separated from the polysaccharide solution by centrifugation at 12,000-14,000 g and filtration steps using depth filters, carbon filters, and 0.45 micron filters. Optionally, the treated solution obtained after Aeroperl treatment may be further passed through a reinforced cellulose membrane bearing quaternary ammonium ligands.

[0109] Process impurities are further removed from the filtered polysaccharide solution by treating it again with a reinforced cellulose membrane bearing a quaternary ammonium ligand. All process impurities are selectively bound to the membrane. The polysaccharide solution is collected as effluent.

[0110] The purified polysaccharide is concentrated and dialyzed using a 100 kDa molecular weight cutoff membrane and saline and water for injection to remove low molecular weight polysaccharides and residual chemicals used in the purification process. The concentrated polysaccharide solution is filtered through a 0.45 micron filter followed by a 0.22 micron filter to remove bioburden. The filtered polysaccharide solution is stored at -20°C.

[0111] The product was characterized using sup.1H-NMR, and the data showed agreement with the chemical structure by assignment of signals assigned to protons of the polysaccharide molecule. The 1H-NMR spectrum showed a series of well-resolved signals (protons from methyl groups) for quantification of the functional groups of the polysaccharide. Multi-angle light scattering technique (MALLS) was used to profile the molecular size distribution of the polysaccharide. The purified polysaccharide conformed to WHO standards.

[0112] Table 3: Impurity levels of capsular polysaccharide from Streptococcus pneumoniae serotype 1 before and after treatment according to the method of the present invention

[0113] [Table 3]

[0114] Table 4: Impurity levels of capsular polysaccharide from Streptococcus pneumoniae serotype 5 before and after treatment according to the method of the present invention

[0115] [Table 4]

[0116] From the data in Tables 3 and 4, it can be inferred that treatment of capsular polysaccharides from Streptococcus pneumoniae serotypes 1 and 5 with reinforced cellulose membranes bearing quaternary ammonium ligands and further exposure to SiO2 is effective in removing impurities well below the permitted regulatory levels, resulting in a superior product.

[0117] advantage The present invention discloses a simple and cost-effective method for purifying capsular polysaccharides without the use of complex and expensive chromatographic methods or toxic reagents such as phenol.

[0118] Additionally, the method of the present invention is robust on a commercial scale, requires only inexpensive process materials, and does not require specialized equipment or hazardous disposal. Furthermore, the method of the present invention helps prevent the formation of high molecular weight polysaccharide aggregates by minimizing inter- and intramolecular bonds. High molecular weight polysaccharide aggregates are difficult to size even at high pressures of about 2000 bar. Using the method of the present invention, capsular polysaccharides can be sized at much lower pressures. For example, capsular polysaccharides can be sized at pressures of approximately 1200 bar or even lower.

Claims

1. 1. A method for purifying capsular polysaccharides, said method comprising the steps of: 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 polysaccharides, proteins, nucleic acids, cell wall components and other impurities; c. Sizing the bacterial cell lysate obtained from step (b) using a homogenizer to obtain a sized crude capsular polysaccharide solution; d. Passing the sized crude capsular polysaccharide obtained in step (c) through a reinforced cellulose membrane having a quaternary ammonium ligand; and e. The solution obtained after passing through the reinforced cellulose membrane with quaternary ammonium in step (d) is treated with silicon dioxide (SiO ２ and isolating the capsular polysaccharide in pure form. A method comprising:

2. 2. The method of claim 1, wherein the bacterium is selected from, but not limited to, the genera Streptococcus, Staphylococcus, Enterococcus, Bacillus, Corynebacterium, Listeria, Erysipelothricus, Clostridium, Haemophilus, Neisseria, or Klebsiella.

3. 3. The method of claim 2, wherein the bacteria is selected from, but not limited to, Streptococcus pneumoniae, Group A Streptococcus, Group B Streptococcus, Group C and G Streptococcus, Staphylococcus aureus, Haemophilus influenzae, Neisseria meningitidis, or Klebsiella pneumoniae.

4. Capsular polysaccharides are 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, 2 2. The method of claim 1, wherein the pneumococcal isolate is isolated from a pneumococcal serotype selected from the group including, but not limited to, 1, 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. 7. The method of claim 6, wherein the capsular polysaccharide is isolated from a Streptococcus pneumoniae serotype selected from serotypes 1 and 5.

6. 10. The method of claim 1, wherein the purified capsular polysaccharide solution obtained from step (e) is further treated with activated carbon to remove colored impurities.

7. 10. The method of claim 1, wherein the purified capsular polysaccharide solution obtained from step (e) is further passed through a reinforced cellulose membrane having quaternary ammonium as a ligand to remove process impurities.

8. 10. The method of claim 1, wherein the purification method is an alcohol-free method and does not use CTAB or other harsh chemicals.

9. 2. The method of claim 1, wherein the reinforced cellulose membrane with quaternary ammonium ligands is equilibrated with a buffer solution until the pH reaches 6.5 to 7.5 and the conductivity reaches 2 to 4 mS / cm.

10. 2. The method of claim 1, wherein the pH of the sized crude capsular polysaccharide solution is adjusted to a range of 6 to 10, and the conductivity of the sized crude capsular polysaccharide solution is adjusted to a range of 1.0 mS / cm to 5.0 mS / cm, and then the solution is passed through a reinforced cellulose membrane having a quaternary ammonium ligand.

11. 2. The method according to claim 1, wherein the sized crude capsular polysaccharide solution obtained from step (c) is passed through a reinforced cellulose membrane at a flow rate ranging from 2 to 10 L / min, preferably from 2 to 5 L / min.

12. SiO used ２ The method of claim 1, wherein the particle size of is in the range of 0.01 μm to 200 μm.

13. SiO ２ The method according to claim 12, wherein the particle size of the powder is preferably in the range of 20 μm to 60 μm.

14. SiO used ２ 2. The method of claim 1, wherein the amount of is in the range of 0.5% to 20% (w / v).

15. SiO ２ The method according to claim 14, wherein the amount of is preferably in the range of 5% to 7% (w / v).

16. In step (e), SiO ２ 2. The method of claim 1, wherein the contacting is carried out at a temperature ranging from 15° C. to 60° C. for a time ranging from 10 minutes to 16 hours.

17. SiO ２ 10. The method of claim 1, wherein is removed using centrifugation at 12,000 to 14,000 g force.

18. 10. The method of claim 1, wherein the fermentation broth is lysed using a lysing agent such as an anionic or cationic detergent.

19. 2. The method of claim 1, wherein the lysing agent is selected from, but not limited to, sodium deoxycholate (DOC), N-lauryl sarcosine (NLS), sodium chenodeoxycholate, and saponin.

20. 10. An immunogenic composition comprising the purified capsular polysaccharide produced according to claim 1 conjugated to a carrier protein selected from diphtheria toxoid, tetanus toxoid, CRM197, PsaA, and PspA.

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