Method for purifying polysaccharides

Abstract

This invention provides a method for purifying polysaccharides from cell lysates. [Solution] A method for purifying polysaccharides comprises: partially purifying a cell lysate containing impurities and polysaccharides to obtain a clarified crude lysate; mixing the clarified crude lysate with a neutralizing solution containing salt to form a neutralized lysate; mixing the neutralized lysate with a precipitate solution containing cetyltrimethylammonium bromide to form a first supernatant and a first precipitate; and separating the first precipitate from the first supernatant, wherein the polysaccharide is located in the first supernatant.

Description

Technical Field

[0001] The present disclosure provides a method for purifying polysaccharides from cells and / or cell lysates. The present disclosure further provides a method for producing polysaccharide vaccines. Vaccines, delivery systems, compositions, and polysaccharides produced by the methods described herein are also provided.

Background Art

[0002] Pathogenic bacteria often have a cell surface capsule coated with polysaccharides. Similarly, pathogenic fungi may have a cell wall containing polysaccharides. During infection, these bacterial and fungal polysaccharides interfere with the immune response by preventing host antibodies from attaching to cells. These polysaccharides [[ID=2(0]]have been found to be effective vaccines against bacterial or fungal pathogens. In certain cases, multiple polysaccharides are conjugated to one vaccine formulation in order to increase protection against multiple serotypes or strains and / or to improve the effectiveness of the vaccine. In addition, polysaccharides are used as non-antigen-specific vaccine adjuvants to enhance the immunogenicity of vaccines.

[0003] The polysaccharides used in vaccine formulations can be isolated from bacteria or fungi after culturing the organisms and then dissolved and purified from unwanted impurities. Typical impurities can include cell proteins, nucleic acids, other cell components, and components of the culture medium. Generally, polysaccharides can be charged, for example, positively or negatively charged, or neutral. The separation of each type of polysaccharide from impurities may require different processes, such as different reagents and concentrations, separation procedures, etc. For example, U.S. Patent No. 5,714,354 describes negatively charged polysaccharides ​​​​​​​​​ Two separate processes are used for the purification of neutral polysaccharides, and each purification process is several This is a multi-day procedure that includes subprocesses. Furthermore, the different processes in each of the separate purification steps The use of reagents can result in reduced yield and quality consistency. Also, different processes The use of this method requires that each step and reagent be evaluated separately during process characterization analysis. This can result in lower efficiency and higher costs, and therefore, for manufacturing compliance reasons. This can increase the burden. Therefore, the purification of polysaccharides is often complicated, time-consuming, and cumbersome. It may be an inefficient effort. [Overview of the project]

[0004] In some embodiments, the present disclosure provides a method for purifying polysaccharides from cell lysates. The method involves (a) partially purifying the cell lysate containing polysaccharides and impurities to obtain a clarified crude solution. (b) obtain the solution, and (b) clarify the crude solution in a solution containing approximately 100 mM to 2 M of salt. (c) Mixing with the solution to form a neutralized solution, and (c) the neutralized solution to approximately Contains 0.1% v / v to approximately 6% v / v of cetyltrimethylammonium bromide (CTAB). (d) Mix with the precipitate solution to form a first supernatant and a first precipitate The process involves separating the urinary components from the first supernatant, and the polysaccharides are substantially located in the first supernatant. ru.

[0005] In some embodiments, partial purification of the cell lysate is performed by precipitation, centrifugation, and filtration. This includes overflow, or a combination thereof. In some embodiments, filtration is deep filtration, contact This includes linear flow filtration (TFF), sterile filtration, or a combination thereof.

[0006] In some embodiments, the neutralization solution contains about 200 mM to about 1 M of salt. In some embodiments, the neutralization solution contains about 250 mM to about 650 mM of salt. In this state, the neutralization solution contains approximately 300 mM to approximately 500 mM of salt. In some embodiments, Salts include NaCl, KCl, CH4Cl, NH4Cl, MgCl2, CaCl2, and Na3 PO4, or a combination thereof. In some embodiments, the neutralizing solution is about 4 Contains 40 mM NaCl.

[0007] In some embodiments, the clarified crude solution in (b) is mixed with the neutralizing solution. This is done simultaneously with or before the filtration step, and the filtration step is before (c) In some embodiments, the mixing in (b) is performed simultaneously with the filtration step. In some embodiments, filtration includes tangential flow filtration (TFF).

[0008] In some embodiments, the precipitated solution contains CTAB at a concentration of approximately 0.1% v / v to approximately 10% v / v. It includes. In some embodiments, the precipitated solution is CT with a concentration of about 0.2% v / v to about 5% v / v. Contains AB. In some embodiments, the precipitated solution is about 0.5% v / v to about 4% v / v Contains CTAB. In some embodiments, the precipitated solution is about 1% v / v to about 3% v / v Includes CTAB.

[0009] In some embodiments, the cell lysate is a positively charged polysaccharide, a neutral polysaccharide, a negatively charged polysaccharide, or This includes combinations thereof. In some embodiments, positively charged polysaccharides, neutral polysaccharides, negatively charged polysaccharides Sugars, or combinations thereof, are essentially present in the first supernatant.

[0010] In some embodiments, the impurities are substantially in the first precipitate. In some embodiments the impurities include polynucleotides. In some embodiments, the polynucleo tide is DNA, RNA, or a combination thereof.

[0011] In some embodiments, mixing in (b), (c), or both is done by shaking , stirring, or pumping. In some embodiments, separating is done by centrifugation, filtration, or a combination thereof.

[0012] In some embodiments, the method further includes subjecting the first supernatant to ultrafiltration, diafiltration, or a combination thereof to produce a retentate and a permeate, wherein the polysaccharide is substantially in the retentate and the CTAB is substantially in the permeate. In some embodiments, the ultrafiltration, diafiltration, or a combination thereof is tangential flow filtration (TFF). 組み合わせに供して、保持液および透過液を生成することをさらに含み、多糖は、実質的 に保持液中にあり、CTABは、実質的に透過液中にある。いくつかの実施形態では、限 外濾過、透析濾過、またはそれらの組み合わせは、接線流濾過(TFF)である。

[0013] In some embodiments, the method further includes subjecting the retentate to carbon filtration. In some embodiments, the method further includes subjecting the retentate to chromatography.

[0014] In some embodiments, the method further includes adding potassium iodide (KI) to the first supernatant to form a second precipitate and a second supernatant, wherein the polysaccharide is substantially in the second supernatant and the CTAB is substantially in the second precipitate. In some embodiments, the method does not include a chromatography step before the addition of KI. 第2の沈殿物および第2の上清を形成することをさらに含み、多糖は、実質的に第2の上 清中にあり、CTABは、実質的に第2の沈殿物中にある。いくつかの実施形態では、方 法は、KIの添加前にクロマトグラフィーステップを含まない。

[0015] In some embodiments, the method centrifuges, filters the second supernatant from the second precipitate, or also This further includes separation by a combination thereof. In some embodiments, The method further includes subjecting the second supernatant to carbon filtration. In some embodiments, the method The method further includes subjecting the second supernatant to chromatography.

[0016] In some embodiments, chromatography is performed on ceramic hydroxyapatite. Type (CHT) chromatography, hydrophobic interaction chromatography (HIC), or a combination thereof. In some embodiments, chromatography is C This includes HT chromatography and HIC. In some embodiments, HIC is performed by This is performed using a chromatography column or membrane containing a nyl group.

[0017] In some embodiments, the method is limited to the following steps after chromatography to concentrate the polysaccharide. Further includes external filtration, diafiltration, or a combination thereof. In some embodiments, Ultrafiltration, dialysfiltration, or a combination thereof, are tangential flow filtration.

[0018] In some embodiments, the method involves the purification of more than 10% of the polysaccharides in the cell lysate. It does not contain the first precipitate. In some embodiments, positively charged polysaccharides, neutral polysaccharides, negatively charged polysaccharides , or combinations thereof, do not precipitate substantially during purification. In some embodiments, Positively charged polysaccharides, neutral polysaccharides, negatively charged polysaccharides, or combinations thereof, are isolated from each other. do not have.

[0019] In some embodiments, more than 90% (v / v) of negatively charged polysaccharides are present in the first supernatant. Negatively charged polysaccharides of less than 10% (v / v) are present in the first precipitate. In some embodiments, More than 98% (v / v) of negatively charged polysaccharides are present in the first supernatant, while less than 2% (v / v) of negatively charged polysaccharides are present. The electropolysaccharide is present in the first precipitate.

[0020] In some embodiments, the yield percentage of polysaccharides is 30% relative to the polysaccharides in the cell lysate. It is greater than %. In some embodiments, the yield percentage of polysaccharides is greater than the percentage of polysaccharides in the cell lysate. In contrast, it is over 40%. In some embodiments, the yield percentage of polysaccharides is greater than that of cell lysates. It is more than 50% of the polysaccharides contained within. In some embodiments, the yield percentage of polysaccharides is The polysaccharide content in the cell lysate is over 60%. In some embodiments, the yield of polysaccharides is The cent is more than 70% of the polysaccharide in the cell lysate. In some embodiments, the polysaccharide The yield percentage is 35% to 80% relative to the polysaccharides in the cell lysate.

[0021] In some embodiments, the cell lysates are derived from bacterial or fungal cells. In that embodiment, the cells are Staphylococcus cells, Streptococcus cells. cus cells, Salmonella cells, Neisseria cells, Mycobacte These are rium cells, or Aureobasidium cells.

[0022] In some embodiments, the cells are Streptococcus cells. In this embodiment, Streptococcus cells are serotypes 1, 2, 3, 4, 5, and 6A. , 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17 These are F, 18C, 19A, 19F, 20A, 22F, 23F, or 33F. In this embodiment, the Streptococcus cells are serotype 3 or 4.

[0023] In some embodiments, the present disclosure provides a method for purifying polysaccharides from cell lysates. The method involves (a) partially purifying the cell lysate containing impurities and polysaccharides to obtain a clarified crude solution. (b) obtain the solution, and (b) clarify the crude solution and add approximately 400 mM sodium chloride (N) (c) Neutralized The dissolved product was mixed with approximately 1.0% v / v cetyltrimethylammonium bromide (CTAB). (d) The first precipitate is formed together with the first supernatant and the first precipitate. (e) Separation from the supernatant, and (e) the first supernatant relative to the second precipitate and the second supernatant. (f) Mix with potassium iodide (KI), and (f) separate the second precipitate from the second supernatant. The polysaccharides, including the phosphate, are substantially located in the second supernatant.

[0024] In some embodiments, the present disclosure provides a method for producing a polysaccharide vaccine, the method is This includes purifying polysaccharides according to the methods provided herein to obtain a polysaccharide vaccine. In several embodiments, the polysaccharides are chitosan, glucan, mannose, inulin, and galactosin. -, galacturonic acid, 2-acetamido-4-amino-2,4,6-trideoxygal Cactose (AATGalp), glucuronic acid, glucose, rhamnose, N-acetylma Mannac, N-acetyl-L-fucosamine, beta -Includes galactofuranose (beta-Galf) or combinations thereof. How many In one embodiment, the polysaccharide is further conjugated with a protein or polypeptide. ru.

[0025] In some embodiments, the present disclosure provides for the purification of a poly(V) by the method provided herein. A vaccine containing sugar is provided. In some embodiments, the polysaccharide is chitosan, glucan, Mannose, inulin, galactose, galacturonic acid, 2-acetamido-4-amino -2,4,6-Trideoxygalactose (AATGalp), glucuronic acid, glucose S, rhamnose, N-acetylmannosamine (ManNAc), N-acetyl-L-fuco Samine (FucNAc), beta-galactofuranose (beta-Galf), or so This includes those combinations.

[0026] In some embodiments, the present disclosure provides for the purification of a poly(V) by the method provided herein. The present invention provides a delivery system containing sugars. In some embodiments, the polysaccharide is pullulan, hyaluronic acid, etc. Ronic acid, alginate, chitosan, dextran, cellulose, or combinations thereof Includes se.

[0027] In some embodiments, the present disclosure provides for the purification of a poly(V) by the method provided herein. The present invention provides a pharmaceutical composition containing sugars. In some embodiments, the polysaccharide comprises chitosan.

[0028] In some embodiments, the present disclosure provides for the purification of a poly(V) by the method provided herein. It provides sugar. [Modes for carrying out the invention]

[0029] This disclosure relates to a method for purifying polysaccharides from cells.

[0030] As used herein, "a" or "an" may mean one or more. When used in writing, the words "a" or "an" are used together with the word "include". When used, it may mean one or more. When used herein, it means "another " or "furthermore" can mean at least a second or more.

[0031] Throughout this application, the term "about" refers to the value and the method used to determine the value. This indicates that the errors may include inherent variations in the device itself, or variations present among the subjects of study. It is used for that purpose. Typically, the term "about" means approximately 1%, 2%, etc., depending on the context. 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% 15%, 16%, 17%, 18%, 19%, or 20%, or 1%, 2%, 3% , 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 1 This means including fluctuations of less than 5%, 16%, 17%, 18%, 19%, or 20%. It tastes good.

[0032] The use of the term "or" in the claims explicitly indicates that it refers only to the substitute. Unless otherwise indicated, it is used to mean "and / or" or alternatives. While these terms are mutually exclusive, this disclosure does not refer to substitutes and the definitions "and / or" only. I support it.

[0033] As used herein, "comprising" and "comp Any variation of "comprising," such as "rise" and "comprises." (or form), "to have" (as well as "have" and "has") (Any variation or form of "having"), "including" (and also "Includes" and "include" and other "Include" words Any variation or form of ", or "containing" (and " Any version of "containing" such as "contains" and "contain" The term (or form) is inclusive or open-ended, and is additional, enumerated. No elements or method steps that are not discussed herein are excluded. This disclosure relates to any method, system, compound, e.g., polysaccharide, and / or composition of any of the methods, systems, compounds, e.g., polysaccharides, and / or compositions of this disclosure. It is intended that this may be carried out. Furthermore, the compositions, systems, and / or Compounds, such as polysaccharides, can be used to achieve the methods of this disclosure.

[0034] The term "for example" and its corresponding abbreviation "for example" e.g.) (whether italicized or not) is used when the listed specific terms are Unless otherwise explicitly stated, it is intended to be limited to the specific examples referenced or cited. This does not mean that these are representative examples and embodiments of the present disclosure.

[0035] As used herein, "between" refers to a range that includes the end of the range. For example, the numbers between x and y include the numbers x and y, as well as the range of x and y. Any number in the specified range is explicitly included.

[0036] As used herein, "polysaccharide" refers to a single molecule linked together by glycosidic bonds. It is a high-molecular-weight carbohydrate compound containing long chains of sugar units. Polysaccharides are highly branched from a linear chain. The structure can vary greatly. Polysaccharides, for example, include starch and glycogen, which are "storage" substances. "Polysaccharides" as well as "structural" polysaccharides such as cellulose, chitin, arabinoxylan, and pectin. It may contain sugars. Polysaccharides can be charged or neutral. A "charged" polysaccharide has a charging group in its molecule. It supports both negatively charged (acidic) and positively charged polysaccharides. Negatively charged polysaccharides are, for example, Positively charged polysaccharides may contain carboxyl groups and / or sulfate ester groups. For example, it can contain protonated free amino groups. Polysaccharides include, for example, Guo Further details can be found in et al., DOI:10.5772 / intechopen.71570. It will be published.

[0037] The polysaccharides of this disclosure can be produced by cells, for example, certain bacteria, typically pathogenic Bacteria produce a thick, mucous membrane-like layer of polysaccharides. This polysaccharide layer is called a "capsule" or "surface capsule." It is sometimes referred to as such, otherwise it would trigger an immune response, thereby destroying the bacteria. It may be a protective mechanism that covers antigenic proteins on the bacterial surface that may be released. Generally, bacteria Capsular polysaccharides are water-soluble, sometimes acidic, and have a molecular weight of approximately 100 to 2000 kDa. Bacterial capsular polysaccharides are typically linear, consisting of regular repeats of 1 to 6 monosaccharides. It can contain subunits. Bacterial capsular polysaccharides exhibit a wide range of structural diversity. In that embodiment, a polysaccharide, for example, a bacterial capsule polysaccharide, is used, for example, for protection against bacteria. , and / or used as an adjuvant in the production of vaccines. Several implementations In this state, polysaccharides are found inside and / or on the surface of cells as described herein, such as bacterial cells. It is present on the surface. In some embodiments, the polysaccharide is present in the cell lysates described herein. It exists.

[0038] As used herein, "precipitation" refers to the formation of a solid in a solution, for example, a liquid solution. This refers to the process of forming a solid, which is called a "precipitate," and the liquid is called the "supernatant." In several embodiments, precipitation is achieved by adding a drug referred to herein as "precipitant". This can be induced. In some embodiments, precipitation can cause one component of the solution to separate from another. Used to separate components. For example, precipitation is used to separate the fine particles described herein. Polysaccharides are separated from undesirable impurities in the cellular lysate, thereby allowing polysaccharides to be extracted from the cell lysate. It can be "purified". Those skilled in the art will understand that the term "purify" means that some of the impurities or It is important to understand that this refers to the complete removal of impurities and does not necessarily mean a composition completely free of impurities. Ro.

[0039] As used herein, "polynucleotide" refers to a covalently linked nucleotide. It means polymer compounds that contain. Polynucleotides are, for example, DNA, RNA, or This includes combinations thereof. In some embodiments, polynucleotides are as described herein. The cells described, for example, bacterial cells, are present inside and / or on the surface. In the embodiment, the polynucleotide is present in the cell lysate described herein. In some embodiments, the polynucleotide is an impurity of the cell lysate described herein. It is considered to be.

[0040] As used herein, "protein" or "polypeptide" refers to any length It refers to the polymer form of amino acids. Proteins include, for example, structural proteins, enzymes, This includes membrane or membrane-related proteins, transporters, receptors, etc. Several implementations Morphologically, the proteins of this disclosure are found inside cells described herein, such as bacterial cells. and / or present on the surface. In some embodiments, the protein is as described herein. It is present in the cell lysates described herein. In some embodiments, the protein is as described herein. It is considered an impurity in the cell lysate described. In some embodiments, proteins Alternatively, polypeptides may be used as vaccine antigens as described herein.

[0041] In some embodiments, the present disclosure uses polysaccharides as polynucleotides and polypeptides. It provides an efficient and simplified method for separating any undesirable impurities. For example, Therefore, this method is independent of the charge of the polysaccharide (e.g., positively charged, negatively charged, and neutral), and is arbitrary. This method can purify polysaccharides of this type. This method also has a step in the polysaccharide purification process. Reduce the number of steps. In some embodiments, the method is, for example, U.S. Patent No. 5,714. As described in No. 354, it does not involve precipitating polysaccharides and then resolubilizing them. Precipitating and resolubilizing sugars ensures complete precipitation and resolubilization of polysaccharides. Because it can be difficult, it may lead to problems with reproducibility. Therefore, this method is currently used It reduces the complexity of the purification process and improves efficiency. For example, vaccines, delivery systems, etc. For manufacturers of pharmaceuticals such as and / or pharmaceutical compositions, the purification of any type of polysaccharide The ability to use a single, streamlined process for the equipment and its maintenance is crucial. Significantly reduce processing and labor costs, use equipment more efficiently, increase manufacturing time, and reduce labor costs. It reduces the cost, simplifies personnel training, reduces the amount of different inspections and certifications, and improves manufacturability. This can improve scaling and other aspects.

[0042] In some embodiments, the same method is used to purify different types of polysaccharides. For example, different polysaccharides differ in charge, size, type of monosaccharide unit, and source (e.g., biological). The serotype and other factors may differ. In some embodiments, different polysaccharides are purified. Using the same method for that purpose is, for example, in a purification process specific to a particular type of polysaccharide. Efficiency is increased by reducing the number of steps. In some embodiments, Using the same method to purify different polysaccharides is, for example, regardless of the type of polysaccharide. Furthermore, consistency can be increased by using the same neutralization and precipitation solutions as well as the same steps. To add. In some embodiments, the same method is used to purify different polysaccharides. This typically involves simplifying process characteristic analysis studies required for the commercialization of new drugs. For example, in order to commercialize a drug product, the manufacturer must verify the drug's manufacturing process. Moreover, this ensures that the manufacturing process provides consistent product quality and that patients are not exposed to risk. We guarantee that this is not the case. Therefore, the same method is used to purify different polysaccharides. In terms of administration methods, even if polysaccharides may be used in different drug products, the test should be performed only once. It needs to be proven, thereby the cost to the manufacturer, and ultimately to the customer, for example, the patient. This will significantly reduce the problem.

[0043] In some embodiments, the present disclosure provides a method for purifying polysaccharides from cell lysates. The method involves (a) partially purifying the cell lysate containing polysaccharides and impurities to obtain a clarified crude solution. (b) obtain the solution, and (b) clarify the crude solution in a solution containing approximately 100 mM to 2 M of salt. (c) Mixing with the solution to form a neutralized solution, and (c) the neutralized solution to approximately Contains 0.1% v / v to approximately 6% v / v of cetyltrimethylammonium bromide (CTAB). (d) Mix with the precipitate solution to form a first supernatant and a first precipitate The process involves separating the urinary components from the first supernatant, and the polysaccharides are substantially located in the first supernatant. ru.

[0044] In some embodiments, cell lysates are obtained by lysing cells. In this embodiment, cells are subjected to mechanical destruction, liquid homogenization, sonication, freeze-thaw, manual grinding, Osmotic dissolution, chemical dissolution (e.g., antibiotics, chelating agents, chaotropic agents, and / or Cells are dissolved using (including the use of solvents) or a combination thereof. The method is known in the relevant technical field, for example, Walker, JM The Pro otein Protocols Handbook, 3 rd Ed. New York (NY): Listed under Springer-Verlag New York, LLC. In some embodiments, the cell lysate comprises a mixture of intracellular and surface components of the cell. In some embodiments, the cell lysate contains impurities and polysaccharides.

[0045] In some embodiments, cell lysates are obtained by lysing bacterial cells. In one embodiment, the bacterial cell is a bacterial cell having a capsule. It has a surface capsule containing polysaccharides. In some embodiments, the polysaccharides on the surface of bacterial cells are fine It generates an immune response from the host cell of the bacteria. In some embodiments, the polysaccharide is used against the bacteria. It is used to produce vaccines. In some embodiments, polysaccharides are used in vaccines. It is used as an adjuvant.

[0046] An example of a bacterium with a surface capsule containing polysaccharides is Staphylococcus. aureus, Streptococcus pneumoniae, Streptococcus Occus agalactiae and other Group A streptococci (GAS) , Streptococcus pyogenes and other Group B streptococcus ci(GBS), Salmonella enterica, Salmonella t yphi, Salmonella paratyphi, Mycobacterium tuberculosis, Neisseria meningitidis, Haem ophilus influenzae, Klebsiella pneumonia, Examples include, but are not limited to, Escherichia coli. In several embodiments, the bacterial cells are Staphylococcus cells, Strepto coccus cells, Salmonella cells, Neisseria cells, or Myc These are bacterium cells. In some embodiments, bacterial cells are Streptococcus. These are ococcus cells. In some embodiments, bacterial cells are Streptoco ccus pneumoniae cells, Streptococcus agalacti These are AE cells, or Streptococcus pyogenes cells. In some embodiments, the bacterial cells are Staphylococcus cells. In terms of morphology, the bacterial cells are Staphylococcus aureus cells. In some embodiments, the bacterial cells are Salmonella cells. Morphologically, the bacterial cell is a Salmonella enterica cell. In some embodiments, the bacterial cell is a Salmonella cell. Bacterial cells include Salmonella enterica cells, for example, Salmon These are Salmonella typhi cells or Salmonella paratyphi cells. In some embodiments, the bacterial cell is a Neisseria cell. In terms of morphology, the bacterial cells are Neisseria meningitidis cells. In some embodiments, the bacterial cells are Mycobacterium cells. In that embodiment, the bacterial cell is Mycobacterium tuberculosi These are S cells. Bacteria contain numerous serotypes corresponding to different antigens on their cell surface. In some embodiments, different serotypes of bacterial organisms have different degrees of toxicity. In some embodiments, the bacterial cells are toxic bacterial cells. In some embodiments, The bacterial cells are Staphylococcus serotypes 1, 2, 5, 8, 336, or NT. These are aureus cells. In some embodiments, bacterial cells are serotype Typhi, Typhimurium, Choleraesuis, Schwarzengrund, Derby, Haifa, Stanley, Newport, Virchow, Para typhi, Singapore, Agona, Panama, Blockley, An atum, Infantis, Enteritidis, Heidelberg, Mue nchen, Javiana, Montevideo, Thompson, or Ora These are Salmonella cells from Nienburg. In some embodiments, bacterial cells The cells are Neisseria meningitidi of serotype A, B, C, W, or Y. These are S cells. In some embodiments, bacterial cells are serotypes 4, 8, 9, or NT. These are Mycobacterium tuberculosis cells. In this state, bacterial cells are classified into serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9A, 9L, 9N, 9V, 10A, 11A, 11B, 11C, 11D, 11F, 12F, 14, 15A , 15B, 15C, 15F, 17F, 18A, 18B, 18C, 18F, 19A, 19B Stre These are ptococcus cells. In some embodiments, Streptococcus The cells are serotype 3 or serotype 4. In some embodiments, Streptoco CCUs cells are serotype 3. In some embodiments, Streptococcus The S cells are serotype 4.

[0047] In some embodiments, the cell lysate is obtained by lysing fungal cells. In some embodiments, the fungal cells have a cell wall containing polysaccharides. In some embodiments, The polysaccharides in the fungal cell wall generate an immune response from the fungal host cell. Several embodiments So, the polysaccharides in fungal cell walls are used to produce vaccines against fungi. How many? In that embodiment, the polysaccharides of the fungal cell wall were used as adjuvants in the vaccine. An example of a fungus with a cell wall containing polysaccharides is Aspergillus fum. igatus, Candida albicans, Cryptococcus neo formans, Histoplasma capsulatum, and Aureob Asidium pullulans is one example, but it is not limited to these. In some embodiments, the fungal cells are Aureobasidium cells. Morphologically, the fungal cell is an Aureobasidium pullulans cell. In some embodiments, the fungal cells are Aureobasidium pullulan s var.pullulans. In some embodiments, the fungal cells are Aur In eobasidium pullulans cells var. melanogenicum Yes. In some embodiments, the fungal cells are Aureobasidium pullu lans MC571 shares, MC573 shares, MC574 shares, MC576 shares, MC711 shares, MC737 shares, MC745 shares, MC767 shares, CBS701.76 shares, or CBS10 5.22 strain. In some embodiments, the fungal cells are Aspergillus f These are umigatus cells. In some embodiments, the fungal cells are Candida cells. These are albicans cells. In some embodiments, serotype A or B candi These are da albicans cells. In some embodiments, fungal cells are Crypt These are ococcus neoformans cells. In some embodiments, fungal cells. Cryptococcus neofor, a serotype A, D, or AD hybrid, is a type of Cryptococcus neofor These are mans cells. In some embodiments, fungal cells are Histoplasma These are capsulatum cells. In some embodiments, serotypes 1, 2, 3, 4, and These are Histoplasma capsulatum cells.

[0048] In some embodiments, the polysaccharides in the cell lysate originate from the cell surface. In some embodiments, the polysaccharide is a neutral polysaccharide. In some embodiments, the polysaccharide is a positively charged polysaccharide. In some embodiments, the polysaccharide is a negatively charged polysaccharide. In some embodiments, The cell lysates contain positively charged polysaccharides, neutral polysaccharides, negatively charged polysaccharides, or combinations thereof. Charged polysaccharides (positively or negatively charged) and neutral polysaccharides are described herein.

[0049] In some embodiments, the polysaccharide is a bacterial surface polysaccharide. The sugar is a fungal cell wall polysaccharide. In some embodiments, the polysaccharide is hyaluronic acid, glucose N-acetylglucosamine, sialic acid, galactose, glucose, N-acetyl Lumannosamine, N-acetylfucosamine, polyribosyl ribitol phosphate (PRP), Galactan, rhamnose, hexuronic acid, mannoocturonic acid, phosphate, cellulose, Pullulan, β-glucan, lectin, dectin-1, dectin-2, galectin-3, ma Glucan, chitin, galactomannan, α-glucan, galactosamine, galactan Toxylomannan, glucuronoxylomannan, chitosan, glucan, mannose, dog Phosphorus, galactose, galacturonic acid, 2-acetamido-4-amino-2,4,6-to Lideoxygalactose (AATGalp), glucuronic acid, glucose, rhamnose, N-acetylmannosamine (ManNAc), N-acetyl-L-fucosamine (FucN Ac), beta-galactofuranose (beta-Galf), or any combination thereof. Includes combinations. In some embodiments, the polysaccharide is chitosan, glucan, mannose, i Nurin, galactose, galacturonic acid, 2-acetamido-4-amino-2,4,6- Trideoxygalactose (AATGalp), glucuronic acid, glucose, rhamnose , N-acetylmannosamine (ManNAc), N-acetyl-L-fucosamine (Fuc NAc), beta-galactofuranose (beta-Galf), or any combination thereof. Includes combinations. Further polysaccharides are, for example, Morris et al., Encycling OPEDIA of Microbiology 3 rd Edition, ed.M. Schaechter, 482-494 (2009), and Snarr et al. Provided in J Fungi 3(3):47(2017).

[0050] In some embodiments, the cell lysate contains polysaccharides and impurities as described herein. In some embodiments, the impurities include intracellular components. In some embodiments, Impurities include polynucleotides, polypeptides, lipids, macromolecules, small molecules, and signaling molecules. Includes offspring, metabolites, membrane components, or combinations thereof. In some embodiments, The pure product contains polynucleotides. In some embodiments, the impurities are DNA, RNA. , or a combination thereof. In some embodiments, the impurities are any of the cells. Contains non-polysaccharide components.

[0051] In some embodiments, one or more impurities are similar to polysaccharides in terms of physical and / or chemical properties. Scientific properties, e.g., similar molecular weight, structural features (e.g., shape, polymerization state, etc.), electric charge, It has density and / or water solubility. In some embodiments, one or more impurities are It has the same charge as polysaccharides. In some embodiments, one or more impurities are positively charged. In some embodiments, one or more impurities are negatively charged. In the application form, one or more impurities are neutral, i.e., have no charge. In the embodiment, the cell lysate contains one or more positively charged impurities, a positively charged polysaccharide, a neutral polysaccharide, and Contains negatively charged polysaccharides. In some embodiments, the cell lysate contains one or more negatively charged impurities. It includes positively charged polysaccharides, neutral polysaccharides, and negatively charged polysaccharides. In some embodiments, it is a cell lysate. It contains one or more neutral impurities, positively charged polysaccharides, neutral polysaccharides, and negatively charged polysaccharides. In this embodiment, the cell lysate contains one or more polynucleotide impurities, positively charged polysaccharides, and neutral It includes polysaccharides and negatively charged polysaccharides. In some embodiments, it includes one or more polynucleotides. The impurities include negatively charged polynucleotide impurities. In some embodiments, negatively charged poly Nucleotide impurities are DNA.

[0052] In some embodiments, one or more impurities are, for example, physical and / or chemical. Due to their similar properties, chromatography, filtration, or centrifugation of cell lysates It is not possible to separate the polysaccharide by separation. In some embodiments, one or more impurities are present. It can be precipitated by a precipitant, while polysaccharides do not form a precipitate in the presence of a precipitant. This separates one or more impurities from the polysaccharide. In an alternative embodiment, one or more The impurities mentioned above remain in the solution in the presence of a precipitating agent while the polysaccharides form a precipitate, and thereafter... Then, one or more impurities are separated from the polysaccharide. In some embodiments, one or more impurities are , containing polynucleotide impurities. In some embodiments, the polynucleotide impurities are It is DNA.

[0053] In some embodiments, cell lysates containing polysaccharides and impurities are clarified crude lysates. The product is partially purified to obtain the substance. In some embodiments, the cell lysate is partially purified. The preparation process includes pH adjustment, precipitation, centrifugation, filtration, or a combination thereof. In some embodiments, precipitation is performed by adjusting the pH so that one or more impurities form a precipitate. This includes the following: In some embodiments, centrifugation is used to separate the soluble fraction of the cell lysate from the insoluble fraction. Separation from the chemical fraction (e.g., precipitate), i.e., centrifugation, clarifies the cell lysates. In some embodiments, filtration is deep filtration, tangential flow filtration (TFF), sterile filtration, and This includes combinations thereof. In some embodiments, filtration includes deep filtration. The person selects the appropriate purification steps and their parameters to obtain a clarified crude product containing polysaccharides. A dissolved product can be obtained. In some embodiments, the clarified crude dissolved product is described in this specification. Contains impurities not removed by the partial purification steps described in the book. Several implementations In its clarified state, the crude solution contains polysaccharides and impurities.

[0054] In some embodiments, the clarified crude solution forms a neutralized solution. It is then mixed with a neutralizing solution containing salt. In some embodiments, the neutralized solution is multiplied It contains sugars and impurities. In some embodiments, the salt is a charged compound, such as a charged polysaccharide. It provides a chemical environment in which it can behave like a neutral polysaccharide. Therefore, several implementations In some forms, the salt neutralizes charged compounds, such as charged polysaccharides. In some embodiments, Neutralization is approximately 100mM to approximately 2M, approximately 150mM to approximately 1.5M, approximately 200mM to approximately 1M, approximately 200mM~Approx. 800mM, Approx. 250mM~Approx. 750mM, Approx. 250mM~Approx. 700mM Approximately 300mM to 600mM, approximately 300mM to 500mM, approximately 400mM to 500 It contains mM, or about 400 mM to about 450 mM of salt. In some embodiments, it is neutralized. The solutions are approximately 100 mM, 150 mM, 200 mM, 225 mM, 250 mM, and 2 75mM, about 300mM, about 325mM, about 350mM, about 375mM, about 400mM, Approximately 425 mm, approximately 450 mm, approximately 475 mm, approximately 500 mm, approximately 550 mm, approximately 600 m M, approx. 650mM, approx. 700mM, approx. 750mM, approx. 800mM, approx. 850mM, approx. 90 0mM, approx. 950mM, approx. 1M, approx. 1.1M, approx. 1.2M, approx. 1.3M, approx. 1.4M, approx. Contains 1.5M, approximately 1.6M, approximately 1.7M, approximately 1.8M, approximately 1.9M, or approximately 2M of salt. .

[0055] In some embodiments, the concentration of salt in the neutralization solution is determined by the number and type of cells, and the clarification process. Volume of crude dissolved product, amount of polysaccharide (predicted, estimated, or measured), amount of impurities (predicted, estimated, (or measured), and one or more additional components that may be present in the clarified crude solution. The selection is based on the following. In some embodiments, the concentration of the salt in the neutralization solution is clarified. It is sufficient to neutralize charged compounds, such as charged polysaccharides, in the crude solution. In the embodiment, the salt does not precipitate the polysaccharide in the substantially neutralized solution.

[0056] In some embodiments, the salt is a chaotropic salt. In some embodiments, The salt is a sodium salt. In some embodiments, the salt is a potassium salt. In one embodiment, the salt is an ammonium salt. In some embodiments, the salt is a halo It is a genide salt. In some embodiments, the salt is a chloride salt. In this state, the salt is a phosphate. In some embodiments, the salt is NaCl, KCl, C H4Cl, NaPO3, NH4Cl, MgCl2, CaCl2, Na2PO4, or so These are combinations. In some embodiments, the neutralizing solution contains NaCl.

[0057] In some embodiments, the neutralization solution is approximately 100 mM to approximately 2 M, and approximately 150 mM to approximately 1. 5M, about 200mM to about 1M, about 200mM to about 800mM, about 250mM to about 750m M, about 250mM to about 700mM, about 300mM to about 600mM, about 300mM to about 50 NaCl at concentrations of 0 mM, approximately 400 mM to 500 mM, or approximately 400 mM to 450 mM Includes. In some embodiments, the neutralizing solution is about 100 mM, about 150 mM, about 200 mM. M, approx. 225mM, approx. 250mM, approx. 275mM, approx. 300mM, approx. 325mM, approx. 35 0mM, approximately 375mM, approximately 400mM, approximately 425mM, approximately 440mM, approximately 450mM, approximately 475mM, approximately 500mM, approximately 550mM, approximately 600mM, approximately 650mM, approximately 700mM Approximately 750mM, approximately 800mM, approximately 850mM, approximately 900mM, approximately 950mM, approximately 1m, Approx. 1.1M, Approx. 1.2M, Approx. 1.3M, Approx. 1.4M, Approx. 1.5M, Approx. 1.6M, Approx. 1.7 It contains M, about 1.8M, about 1.9M, or about 2M of NaCl. In some embodiments, The neutralization solution contains approximately 440 mM NaCl. In some embodiments, the neutralization solution contains The amount of NaCl neutralizes the charged compounds, such as charged polysaccharides, in the clarified crude solution. This is sufficient. In some embodiments, the NaCl in the neutralization solution is substantially neutralized. The solution does not allow polysaccharides to precipitate.

[0058] In some embodiments, a clarified crude solution is used to form a neutralized solution. Mixing with the neutralizing solution is performed simultaneously with the filtration step. In some embodiments, Mixing the clarified crude solution with the neutralizing solution to form a neutralized solution is This is performed before the filtration step. In some embodiments, filtration is performed by tangential flow filtration (TFF). Includes. In some embodiments, the TFF buffer includes a neutralizing solution, which is then mixed. The filtration step is performed simultaneously. In some embodiments, the filtration step is performed on a neutralized This is done before mixing the dissolved substance with the precipitate solution.

[0059] In some embodiments, the neutralized solution is mixed with a precipitate solution containing a precipitating agent. In some embodiments, the precipitated solution is cetyltrimethylammonium bromide (CTAB) It includes. In some embodiments, the precipitating agent is CTAB. In some embodiments, Polysaccharides substantially do not form precipitates in the precipitated solution. In some embodiments, one or more The impurities mentioned above are substantially present in the first precipitate in the precipitated solution. In some embodiments, Polynucleotide impurities, when the precipitated solution is mixed with the neutralized soluble product, form the first precipitate. The polysaccharides in the neutralized dissolved product are soluble when the precipitated solution is mixed with the cell lysates. It remains in its original state. Therefore, in some embodiments, one or more impurities are present in the first precipitate. It is located inside. In some embodiments, the polysaccharide is substantially located in the first supernatant. In that embodiment, positively charged polysaccharides, neutral polysaccharides, negatively charged polysaccharides, or combinations thereof are It is present in the first supernatant. In some embodiments, one or more impurities are negatively charged polynucleotides. Contains ocidal impurities. In some embodiments, negatively charged polynucleotide impurities are DN It is A.

[0060] Those skilled in the art will understand that, during the precipitation of impurities, 100% of a given impurity does not usually precipitate. Therefore, the term "substantially" refers to the precipitation of impurities. The amount of a given impurity that the company expects to precipitate, for example, more than 60% of the given impurity, This refers to percentages exceeding approximately 70%, 80%, 90%, or 95%.

[0061] When CTAB is added to the neutralized solution described herein, polynucleotides (e.g.) For example, it can selectively precipitate DNA, while polysaccharides (for example, positively charged, negatively charged) can be selectively precipitated. It was found that neutral polysaccharides (or combinations thereof) remain in the solution. Theoretically... Without being constrained, CTAB precipitates negatively charged DNA, while, as described herein, As such, charged polysaccharides (e.g., negatively charged polysaccharides) are neutralized, and the resulting solution contains neutral polysaccharides. Because of this behavior, polysaccharides of any charge (including negatively charged polysaccharides) remain substantially in the solution. This is thought to be possible. "Substantially in solution" means approximately 50% of the polysaccharides in the dissolved cells. Very, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, about 8 More than 5%, more than about 90%, more than about 91%, more than about 92%, more than about 93%, more than about 94%, more than about 95%, Approximately 96% or more, approximately 97% or more, approximately 98% or more, approximately 99% or more, or approximately 100% are the first and / Or it means that it is in the second supernatant. Therefore, this disclosure separates polysaccharides from impurities. It provides improved efficiency and simplicity for doing so.

[0062] In some embodiments, the precipitated solution is CTAB greater than 0.1% v / v, or 0.1% It contains v / v ~ 20% v / v CTAB. In some embodiments, the precipitated solution is about 0. CTAB from 1%v / v to approximately 10%v / v, CTAB from approximately 0.1%v / v to approximately 6%v / v, Alternatively, CTAB of approximately 0.2%v / v to approximately 5%v / v, or approximately 0.5%v / v to approximately 4%v CTAB of / v, or CTAB of approximately 1%v / v to approximately 3%v / v, or approximately 1.5%v / Includes CTABs of approximately 2.5% v / v, or CTABs of approximately 1% v / v to 2% v / v. In some embodiments, the precipitated solution is about 0.1% v / v, about 0.2% v / v, about 0. 3%v / v, approx. 0.4%v / v, approx. 0.5%v / v, approx. 0.6%v / v, approx. 0.7%v / v, approx. 0.8%v / v, approx. 0.9%v / v, approx. 1%v / v, approx. 1.1%v / v, approx. 1.2 %v / v, approx. 1.3%v / v, approx. 1.4%v / v, approx. 1.5%v / v, approx. 1.6%v / v , approx. 1.7%v / v, approx. 1.8%v / v, approx. 1.9%v / v, approx. 2%v / v, approx. 2.1% v / v, approx. 2.2%v / v, approx. 2.3%v / v, approx. 2.4%v / v, approx. 2.5%v / v, Approx. 2.6%v / v, approx. 2.7%v / v, approx. 2.8%v / v, approx. 2.9%v / v, approx. 3.0 %v / v, approx. 3.1%v / v, approx. 3.2%v / v, approx. 3.3%v / v, approx. 3.4%v / v , about 3.5%v / v, about 3.6%v / v, about 3.7%v / v, about 3.8%v / v, about 3. Contains 9% v / v, or approximately 4% v / v of CTAB.

[0063] In some embodiments, the concentration of CTAB in the precipitate solution depends on the number and type of cells. Volume of dissolved solution, amount of polysaccharide (predicted, estimated, or measured), amount of impurities (predicted, estimated) (or measured), and one or more of the additional components that may be present in the neutralized solution. The selection is based on the following. In some embodiments, the amount of CTAB in the precipitate solution is determined by the amount of impurities. Sufficient for precipitation. In some embodiments, the amount of CTAB in the precipitated solution is D Sufficient to precipitate NA. In some embodiments, CTAB in the precipitate solution is The polysaccharides are substantially not precipitated. In some embodiments, the polysaccharides are substantially in the first supernatant. It is located in some embodiments. Positively charged polysaccharides, neutral polysaccharides, negatively charged polysaccharides, or those The combination is substantially located in the first supernatant. In some embodiments, impurities, e.g. For example, DNA is essentially present in the first precipitate.

[0064] In some embodiments, mixing with the precipitated solution of the neutralized dissolve is done by shaking, stirring, pumping, etc. This is done by ping, or a combination thereof. In some embodiments, neutralization Mixing the dissolved solution with the precipitated solution promotes the formation of the precipitate. In some embodiments, Mixing with the neutralized dissolved precipitate solution improves the separation of the first precipitate from the first supernatant. ru.

[0065] In some embodiments, positively charged polysaccharides, negatively charged polysaccharides, neutral polysaccharides, or combinations thereof The method involves precipitation of polynucleotide impurities without a chromatography step. Renucleotide impurities, for example, can be removed from DNA. Therefore, several In one embodiment, the method involves chromatography before separating the first precipitate from the first supernatant. - Does not include a step. A non-limiting example of chromatography is anion exchange chromatography. Mathematics, cation exchange chromatography, hydrophobic interaction chromatography Examples include affinity chromatography and size exclusion chromatography.

[0066] In some embodiments, the first precipitate is mixed with the precipitate solution of the neutralized dissolved product after It is separated from the first supernatant. In some embodiments, separation is achieved by centrifugation, filtration. This is done by decanting, decanting, or a combination thereof. In some embodiments, Separation is carried out by centrifugation or filtration. Those skilled in the art will know the parameters of centrifugation. For example, speed and duration, or filtration techniques, such as vacuum filtration, gravity filtration, or These are combinations of them, or decanting devices, for example, container type and decanting angle. By selecting, the first precipitate and the first supernatant can be properly separated.

[0067] In some embodiments, CTAB and polysaccharides are present in the first supernatant after mixing and separation. Yes. In the downstream processing of polysaccharides provided herein, the presence of CTAB is undesirable. In some cases, the method involves a first precipitate and a first supernatant. The further step involves removing CTAB from the first supernatant after separation. In some embodiments, The first supernatant is subjected to ultrafiltration, diafiltration, or to produce retaining fluid and permeate. In these combinations, the polysaccharide is in the holding solution, and CTAB is in the permeate. Generally, ultrafiltration and / or diafiltration (UF / DF) involves passing through a UF / DF membrane. A "permeate" containing materials that can pass through the UF / DF membrane, and a " A "retention solution" is generated. In some embodiments, the UF / DF membrane pore size is such that CTAB The method is selected to allow passage through the membrane, while the polysaccharide is selected to prevent passage through the membrane. In such embodiments, the method is selected to allow passage through the membrane. CTAB is removed from the first supernatant without a second precipitation step, thereby purifying the polysaccharide. It offers the advantage of simplifying the process. In some embodiments, positively charged polysaccharides, neutral polysaccharides, Negatively charged polysaccharides, or combinations thereof, are substantially located in the holding solution.

[0068] One or more undesirable impurities are generally removed from the cell lysate by precipitation in the precipitation solution. Although removed, additional soluble impurities remain in the first supernatant and / or the holding solution containing polysaccharides. These may remain. These additional impurities include, for example, proteins, peptides, and proteins. This may include sexual compounds, as well as small molecules such as signaling molecules or metabolites. Yes, it is possible. In some embodiments, the method involves removing one or more impurities from the holding liquid. The method further includes subjecting the retaining liquid to filtration to remove one or more non- Further includes removing pure substances. In some embodiments, one or more impurities are tan. Contains powder. In some embodiments, filtration is performed by filtering them into a filter or filter resin. Differential adsorption separates one or more impurities from the polysaccharide. In some embodiments, filtration The process separates one or more impurities from the polysaccharide based on molecular weight. In some embodiments, Filtration includes carbon filtration. A non-limiting example of a carbon filter is CUNO NORI Examples include T (registered trademark), ZETA PLUS (trademark), and VAPLOCK (trademark). ru.

[0069] In some embodiments, the method further involves subjecting the retention solution to chromatography. Includes. In some embodiments, the retaining liquid is filtered, for example, carbon filtration followed by chromatography. It is subjected to . In some embodiments, chromatography is performed on one of the second supernatants. Remove the impurities mentioned above. In some embodiments, the impurities include proteins. In several embodiments, chromatography involves anion exchange, cation exchange, and hydrophobic interaction. This includes action, affinity, size exclusion chromatography, or a combination thereof. In several embodiments, chromatography is performed using a ceramic hydroxyapatite type (CHT) chromatography, hydrophobic interaction chromatography (HIC), or This includes combinations thereof. In some embodiments, chromatography is, for example, CHT chromatograph using CHT resin from BIORAD LABORATORIES This is a graph. CHT resins are typically ion exchange (e.g., cation exchange). This includes combinations of affinity (e.g., calcium affinity) resins.

[0070] In some embodiments, the method involves a holding solution in at least two chromatographic stages. This further includes subjecting the sample to chromatography. Therefore, in some embodiments, chromatography is performed. This includes CHT chromatography and HIC. In some embodiments, the method is The retention solution is subjected to CHT chromatography, followed by hydrophobic interaction chromatography (HIC). This includes subjecting the product to a chromatography assay. Two consecutive chromatography steps are performed to obtain the desired polysaccharide. An unexpected finding was that it advantageously improved the removal of undesirable impurities from the contained solution. Examples of ligands for HIC resins include phenyl groups, octyl groups, and butyl groups. However, it is not limited to these. Suitable HIC resins include, for example, SARTORI SARTOBIND film from US, G-SEP agar from G-BIOSCIENCES Examples include high-speed flow resins, as well as CAPTO and SEPHAROSE resins from GE. In some embodiments, HIC is performed using a phenyl film resin.

[0071] In some embodiments, carbon filtration is performed using the same equipment as chromatography. This is done. For example, the retaining liquid is filtered through a carbon filtration column, and then the carbon filtered retaining liquid is collected. Then, without any intermediate steps, the chromatography passes through the chromatography column. It can be made to happen.

[0072] In some embodiments, the first supernatant containing CTAB and polysaccharides is combined with the second precipitate solution. They are mixed. In some embodiments, the second precipitate solution contains a different precipitant than the precipitate solution. Includes. In some embodiments, the first supernatant is mixed with potassium iodide (KI), A second precipitate and a second supernatant are formed. In some embodiments, KI is a polysaccharide. It does not form a precipitate in CTAB. In some embodiments, the polysaccharide is substantially second In the supernatant, CTAB is substantially present in the second precipitate. In some embodiments, Positively charged polysaccharides, neutral polysaccharides, negatively charged polysaccharides, or combinations thereof are essentially a second supernatant. It is located in the middle. In some embodiments, the second precipitate solution is about 0.1% v / v to about 6% v / v KI, or approximately 0.2% v / v to approximately 5% v / v KI, or approximately 0.5% v / v KI of approximately 4% v / v, or approximately 1% v / v to approximately 3% v / v, or approximately 1.5% v KI of approximately 2.5% v / v, or approximately 1% v / v to approximately 2% v / v, or approximately 0 It contains KI at a concentration of 0.5% v / v to approximately 1% v / v. In some embodiments, the amount of KI is 1 The selection is based on the amount of CTAB in the supernatant. It is essentially not any polysaccharide, and is essentially The appropriate concentration of KI for proper precipitate formation in all CTABs is known to those skilled in the art. You can choose.

[0073] In some embodiments, the method involves chromatography before adding KI to the first supernatant. The method does not include a step. In some embodiments, the method involves anion exchange before the addition of KI. Chromatography, cation exchange chromatography, hydrophobic interaction chromatography - Does not include affinity chromatography or size exclusion chromatography steps. i. In some embodiments, the method involves anion exchange chromatography before the addition of KI. No steps are included. No chromatography step is required before the addition of KI. In other words, by removing CTAB by precipitation, this method further enhances the polysaccharide purification process. It can be simplified.

[0074] In some embodiments, the method further separates the second supernatant from the second precipitate. It is included in. In some embodiments, the second supernatant is separated from the second precipitate by centrifugation. Includes separation, filtration, decanting, or a combination thereof. Separating precipitate and supernatant. The method is known in the art and is provided herein. In some embodiments, the second The supernatant is separated from the second precipitate by carbon filtration. Carbon filtration is further described herein. This is described herein. In some embodiments, the second supernatant is the retaining liquid described herein and In a similar manner, the material is subjected to chromatography after carbon filtration. In some embodiments, Chromatography involves anion exchange, cation exchange, hydrophobic interactions, affinity, and size. Exclusion chromatography, or a combination thereof. In some embodiments, Chromatography is performed using ceramic hydroxyapatite type (CHT) chromatography. Raffie, hydrophobic interaction chromatography (HIC), or a combination thereof Includes. In some embodiments, the method involves the second supernatant being subjected to CHT chromatography, followed by This includes subjecting the CHT chromatography resin and HIC ligand to HIC. Examples of resins are provided herein. In some embodiments, the HIC is phenyl This is carried out using a membrane resin. In some embodiments, carbon filtration is described herein. This is performed using the same equipment as chromatography.

[0075] In some embodiments, the polysaccharide is concentrated. In some embodiments, the concentration is Chromatography of the retaining solution or second supernatant as described in the specification, e.g., CHT This is performed after chromatography and HIC. The enrichment method is known in the field, for example This includes rotary evaporation, distillation, and ultrafiltration / dialysis filtration, such as tangential flow filtration. In the embodiments, concentration includes ultrafiltration, diafiltration, or a combination thereof. In some embodiments, concentration is performed, for example, to form concentrated polysaccharides. This includes tangential flow filtration. In some embodiments, the concentrated polysaccharide is substantially free of impurities. stomach.

[0076] This method removes undesirable impurities, such as polynucleotides or proteins. This advantageously enables the effective separation of sugars. In some embodiments, polysaccharides are separated through the method. It remains substantially in solution. As described herein, “substantially in solution” means that it remains substantially in solution. Over 50%, over 55%, over 60%, over 65%, over 70%, and over 75% of the polysaccharides in the solution. More than %, more than about 80%, more than about 85%, more than about 90%, more than about 91%, more than about 92%, more than about 93%, about Over 94%, approximately over 95%, approximately over 96%, approximately over 97%, approximately over 98%, approximately over 99%, or approximately 1 00% means that it is present in the first supernatant, the second supernatant, and / or the retaining solution.

[0077] In some embodiments, this method involves removing more than 10% and 15% of the polysaccharides in the cell lysate during purification. Contains over 20%, over 25%, over 35%, approximately over 40%, approximately over 45%, or approximately over 50%. It does not contain precipitates. In some embodiments, positively charged polysaccharides, neutral polysaccharides, negatively charged polysaccharides, Or, combinations thereof, do not precipitate substantially during purification. This refers to less than 1%, less than 2%, less than 3%, less than 4%, and less than 5% of all polysaccharides in the cell lysate. , less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, or Less than 40% are located in the first precipitate, the second precipitate, and / or the permeate. It means.

[0078] In some embodiments, positively charged polysaccharides, neutral polysaccharides, negatively charged polysaccharides, or combinations thereof The waste product is not substantially located in the first supernatant, the second supernatant, and / or the retaining solution. In some embodiments, positively charged polysaccharides, neutral polysaccharides, and negatively charged polysaccharides are used in the method, and are not combined with each other. They won't let go.

[0079] In some embodiments, "substantially a first supernatant, a second supernatant, and / or a retaining liquid." The polysaccharides located within the cell lysate make up more than 60%, more than 65%, and more than 70% of all polysaccharides. Over 75%, over 80%, over 85%, over 90%, over 95%, over 96%, over 97%, over 98%, Or more than 99% are located in the first supernatant, the second supernatant, and / or the retaining solution. To refer to. Therefore, in some embodiments, the phrase "substantially located in the first supernatant" In this case, more than 60% (v / v) of negatively charged polysaccharides are present in the first supernatant, and less than 40% (v / v) of negatively charged polysaccharides. This means that the negatively charged polysaccharide is present in the first precipitate. In some embodiments, 65% Negatively charged polysaccharides greater than (v / v) are present in the first supernatant, while negatively charged polysaccharides less than 35% (v / v) are present. It is present in the first precipitate. In some embodiments, more than 70% (v / v) of negatively charged polysaccharides. It is present in the first supernatant, and less than 30% (v / v) of negatively charged polysaccharides are present in the first precipitate. In some embodiments, more than 75% (v / v) of negatively charged polysaccharides are present in the first supernatant. Negatively charged polysaccharides of less than 25% (v / v) are present in the first precipitate. In some embodiments, More than 80% (v / v) of negatively charged polysaccharides are present in the first supernatant, while less than 20% (v / v) of negatively charged polysaccharides are present. The charged polysaccharide is present in the first precipitate. In some embodiments, more than 85% (v / v) of negative charge is present. Charged polysaccharides are present in the first supernatant, and negatively charged polysaccharides (less than 15% (v / v)) are present in the first precipitate. It's inside.

[0080] In some embodiments, more than 90% (v / v) of negatively charged polysaccharides are present in the first supernatant. Negatively charged polysaccharides of less than 10% (v / v) are present in the first precipitate. In some embodiments, More than 95% (v / v) of negatively charged polysaccharides are present in the first supernatant, while less than 5% (v / v) of negatively charged polysaccharides are present. The electropolysaccharide is present in the first precipitate. In some embodiments, there is a negative band greater than 96% (v / v). Electropolysaccharides are present in the first supernatant, and negatively charged polysaccharides (less than 4% (v / v)) are present in the first precipitate. Yes. In some embodiments, more than 97% (v / v) of negatively charged polysaccharides are present in the first supernatant. Furthermore, negatively charged polysaccharides of less than 3% (v / v) are present in the first precipitate. In some embodiments, More than 98% (v / v) of negatively charged polysaccharides are present in the first supernatant, while less than 2% (v / v) of negatively charged polysaccharides are present. The charged polysaccharide is present in the first precipitate. In some embodiments, more than 99% (v / v) negative Charged polysaccharides are present in the first supernatant, while negatively charged polysaccharides (less than 1% (v / v)) are present in the first precipitate. It is located there.

[0081] In some embodiments, more than 60% (v / v) of positively charged polysaccharides are present in the first supernatant. Positively charged polysaccharides of less than 40% (v / v) are present in the first precipitate. In some embodiments, Positively charged polysaccharides exceeding 65% (v / v) are present in the first supernatant, while positively charged polysaccharides less than 35% (v / v) are present. The charged polysaccharide is present in the first precipitate. In some embodiments, more than 70% (v / v) positive Charged polysaccharides are present in the first supernatant, and positively charged polysaccharides at less than 30% (v / v) are present in the first precipitate. It is located inside. In some embodiments, more than 75% (v / v) of positively charged polysaccharides are present in the first supernatant. Positively charged polysaccharides of less than 25% (v / v) are present in the first precipitate. Several implementations Morphologically, more than 80% (v / v) of positively charged polysaccharides are present in the first supernatant, and 20% (v / v) Positively charged polysaccharides of less than 85% (v / v) are present in the first precipitate. In some embodiments, 85% (v / v) Positively charged polysaccharides exceeding 15% (v / v) are present in the first supernatant, and positively charged polysaccharides less than 15% (v / v) are present in the first supernatant. It is found in the precipitate.

[0082] In some embodiments, more than 90% (v / v) of positively charged polysaccharides are present in the first supernatant. Positively charged polysaccharides of less than 10% (v / v) are present in the first precipitate. In some embodiments, Positively charged polysaccharides exceeding 95% (v / v) are present in the first supernatant, while positively charged polysaccharides exceeding 5% (v / v) are present in the first supernatant. The electropolysaccharide is present in the first precipitate. In some embodiments, a band greater than 96% (v / v) is present. Electropolysaccharides are present in the first supernatant, and positively charged polysaccharides are present in the first precipitate at a concentration of less than 4% (v / v). Yes. In some embodiments, more than 97% (v / v) of positively charged polysaccharides are present in the first supernatant. Furthermore, positively charged polysaccharides of less than 3% (v / v) are present in the first precipitate. In some embodiments, More than 98% (v / v) of positively charged polysaccharides are present in the first supernatant, and less than 2% (v / v) of positively charged polysaccharides are present. The charged polysaccharide is present in the first precipitate. In some embodiments, more than 99% (v / v) positive Charged polysaccharides are present in the first supernatant, and positively charged polysaccharides are present in the first precipitate at a concentration of less than 1% (v / v). It is located there.

[0083] In some embodiments, more than 60% (v / v) of neutral polysaccharides are present in the first supernatant, and 4 Neutral polysaccharides less than 0% (v / v) are present in the first precipitate. In some embodiments, 6 Neutral polysaccharides exceeding 5% (v / v) are present in the first supernatant, while neutral polysaccharides less than 35% (v / v) are present. It is present in the first precipitate. In some embodiments, more than 70% (v / v) of neutral polysaccharides Neutral polysaccharides are present in the first supernatant, and less than 30% (v / v) are present in the first precipitate. In several embodiments, more than 75% (v / v) of neutral polysaccharides are present in the first supernatant, and 25% Neutral polysaccharides less than (v / v) are present in the first precipitate. In some embodiments, 80% Neutral polysaccharides greater than (v / v) are present in the first supernatant, and neutral polysaccharides less than 20% (v / v) are present. It is present in the first precipitate. In some embodiments, more than 85% (v / v) of neutral polysaccharides is present. Neutral polysaccharides present in the supernatant of 1, and less than 15% (v / v), are found in the first precipitate.

[0084] In some embodiments, more than 90% (v / v) of the neutral polysaccharide is in the first supernatant, and less than 1 0% (v / v) of the neutral polysaccharide is in the first precipitate. In some embodiments, 9 more than 5% (v / v) of the neutral polysaccharide is in the first supernatant, and less than 5% (v / v) of the neutral polysaccharide is , in the first precipitate. In some embodiments, more than 96% (v / v) of the neutral polysaccharide is in the first supernatant, and less than 4% (v / v) of the neutral polysaccharide is in the first precipitate. In some embodiments, more than 97% (v / v) of the neutral polysaccharide is in the first supernatant, and less than 3% (v / v) of the neutral polysaccharide is in the first precipitate. In some embodiments, more than 98% (v / v) of the neutral polysaccharide is in the first supernatant, and less than 2% (v / v) of the neutral polysaccharide is in the first precipitate . In some embodiments, more than 99% (v / v) of the neutral polysaccharide is in the first supernatant[[ID=·19]] and less than 1% (v / v) of the neutral polysaccharide is in the first precipitate.

[0085] In some embodiments, the method advantageously provides a high percentage yield of polysaccharide from the cell lysate . "Percent yield" or "recovery percent" refers to the amount of polysaccharide isolated after purification relative to the total amount of polysaccharide present in the cell lysate before purification . In some embodiments, the percent yield of polysaccharide in the retention solution, the second supernatant, and / or the concentrated polysaccharide is more than 50% relative to the polysaccharide in the cell lysate. In some embodiments, the percent yield of polysaccharide in the retention solution, the second supernatant , and / or the concentrated polysaccharide is more than 5 0% relative to the polysaccharide in the cell lysate. In some embodiments, the percent yield of polysaccharide in the retention solution, the second supernatant, and / or the concentrated polysaccharide is more than 55% relative to the polysaccharide in the cell lysate. In some embodiments, the percent yield of polysaccharide in the retention solution, the second supernatant, and / or the concentrated polysaccharide In the embodiment, the yield percentage of polysaccharides in the retaining solution, the second supernatant, and / or the concentrated polysaccharide. It is more than 60% of the polysaccharides in the cell lysate. In some embodiments, the retaining solution, The yield percentage of polysaccharides in the supernatant and / or concentrated polysaccharide is equal to the polysaccharide in the cell lysate. In contrast, it is over 65%.

[0086] In some embodiments, the retaining solution, the second supernatant, and / or the polysaccharide in the concentrated polysaccharide are used. The percentage is greater than 70% relative to the polysaccharides in the cell lysate. In some embodiments, The yield percentage of polysaccharides in the retention solution, the second supernatant, and / or concentrated polysaccharide is the cell lysis. It is more than 75% of the polysaccharide in the substance. In some embodiments, a retaining solution, a second supernatant, The yield percentage of polysaccharides in concentrated polysaccharides is 80% of the polysaccharides in cell lysates. It is super. In some embodiments, in the retaining liquid, the second supernatant, and / or concentrated polysaccharide The yield percentage of polysaccharides is over 85% relative to the polysaccharides in the cell lysates. Several implementations In terms of form, the yield percentage of polysaccharides in the retaining solution, the second supernatant, and / or concentrated polysaccharide is It is more than 90% of the polysaccharides in the cell lysate. In some embodiments, the retaining solution, second The yield percentage of polysaccharides in the supernatant and / or concentrated polysaccharide is relative to the polysaccharides in the cell lysate. The concentration is over 95%. In some embodiments, a retaining solution, a second supernatant, and / or concentrated solution are used. The yield percentage of polysaccharides in polysaccharides is 35% to 80% relative to the polysaccharides in the cell lysate. In some embodiments, the polysaccharides in the retaining solution, the second supernatant, and / or the concentrated polysaccharide are positive Includes charged polysaccharides, neutral polysaccharides, negatively charged polysaccharides, or combinations thereof.

[0087] In some embodiments, this disclosure relates to the cell lysates described herein and to the herein The present invention provides a composition comprising the neutralizing solution described herein. In some embodiments, the present invention provides A composition comprising, a cell lysate as described herein and a precipitate solution as described herein. The product is provided. In some embodiments, the cell lysate is derived from bacterial or fungal cells. In some embodiments, the neutralizing solution contains NaCl. The precipitate solution contains CTAB. In some embodiments, the present disclosure describes the precipitate and supernatant. The present invention provides a composition containing the following: the precipitate contains the impurities described herein, and the supernatant contains the following Contains polysaccharides as described in the book. In some embodiments, impurities are polynucleotides, e.g. For example, this includes DNA, proteins, or combinations thereof. In some embodiments, this includes DNA, proteins, or combinations thereof. The supernatant consists of positively charged polysaccharides, neutral polysaccharides, negatively charged polysaccharides, or combinations thereof, NaCl, and includes CTAB.

[0088] In some embodiments, the present disclosure relates to a second composition comprising a second precipitate and a second supernatant. The substance is provided, the second precipitate contains CTAB, and the second supernatant contains the various substances described herein. Contains sugar. In some embodiments, the second supernatant contains positively charged polysaccharides, neutral polysaccharides, and negatively charged polysaccharides. , or combinations thereof, including NaCl and KI.

[0089] In some embodiments, the present disclosure provides a method for producing a polysaccharide vaccine, the method is The process includes purifying polysaccharides from cell lysates, and the purification process involves (a) removing polysaccharides and impurities. (b) Clarification The obtained crude lysate is mixed with a neutralization solution containing about 100 mM to about 2 M of salt to form a neutralized lysate, (c) the neutralized lysate is mixed with a precipitation solution containing about 0.1% v / v to about 6% v / v of cetyltrimethylammonium bromide (CTAB) to form a first supernatant and a first precipitate, and (d) separating the first precipitate from the first supernatant, wherein the polysaccharide is substantially located in the first supernatant.

[0090] In some embodiments, the polysaccharide comprises chitosan, glucan, mannose, inulin, galactose, galacturonic acid, 2-acetamido-4-amino-2,4,6-trideoxy galactose (AATGalp), glucuronic acid, glucose, rhamnose, N-acetyl mannosamine (ManNAc), N-acetyl-L-fucosamine (FucNAc), beta -galactofuranose (beta-Galf), or a combination thereof. In some embodiments, the polysaccharide is further conjugated with a protein or polypeptide. Methods for conjugating a polysaccharide to a protein or polypeptide are known in the art, such as, for example, Guo et al., Methods Mol Med 6 6:49-54 (2001), Turner et al., Syn Sys Biol 2(1):49-58 (2017), and U.S. Patent Application Publication No. 2007 / 01410 84.

[0091] In some embodiments, the protein or polypeptide is derived from the same cell as the polysaccharide. In some embodiments, the protein or polypeptide is derived from the same organism as the polysaccharide. In some embodiments, the protein or polypeptide is derived from a different organism than the polysaccharide.​​​​ It originates from. In some embodiments, the organism is a bacterial organism. In some embodiments The organism is a fungal organism. In some embodiments, the protein or polypeptide is , generating an immune response in the host cells of organisms from which the protein or polypeptide originates. In some embodiments, the protein or polypeptide is the vaccine antigen.

[0092] In some embodiments, the present disclosure provides for the purification of a poly(V) by the method provided herein. The present invention provides a vaccine containing sugar. In some embodiments, the polysaccharide is used as an adjuvant in the vaccine. In some embodiments, polysaccharides are the antigens of the vaccine. In this form, the vaccine contains polysaccharides conjugated with different antigens. In this context, polysaccharides are proteins or polypeptides as described herein, for example, protein It is conjugated with a protein or polypeptide vaccine antigen. In some embodiments, Polysaccharides conjugated to proteins or polypeptides are polysaccharides or proteins Alternatively, a stronger immune response from host cells compared to an immune response to polypeptides alone. It generates an answer, thereby providing improved protection to the host cell for the organism. In that embodiment, the polysaccharide-containing vaccine provided herein is a polysaccharide-free vaccine Compared to [another method], it provides improved protection to host cells for organisms. Several implementations In this context, vaccines containing polysaccharide vaccine adjuvants provided herein are polysaccharide vaccines Compared to vaccines without adjuvants, improved retention of host cells in organisms. It provides protection. In some embodiments, the polysaccharide is chitosan, glucan, mannose, i Nurin, galactose, galacturonic acid, 2-acetamido-4-amino-2,4,6- Trideoxygalactose (AATGalp), glucuronic acid, glucose, rhamnose , N-acetylmannosamine (ManNAc), N-acetyl-L-fucosamine (Fuc NaAc), beta-galactofuranose (beta-Galf), or a combination thereof This includes [specific example]. A non-exclusive example of a polysaccharide vaccine is Pfizer / Baxter Int PREVENAR 13 (registered trademark) from international, MENINGITEC (Registered Trademark), and NEISVAC-C (Registered Trademark), and GlaxoSmith MENVEO (registered trademark) and MENJUGATE (registered trademark) from Klin are cited. (It can be done).

[0093] In some embodiments, the present disclosure provides for the purification of a poly(V) by the method provided herein. Provides a delivery system containing sugar. Where used herein, “delivery system” means, for example, For example, formulations that enable and / or facilitate the introduction of substances into the body and / or This refers to a device. In some embodiments, the delivery system delivers therapeutic substances into the body. In some embodiments, the delivery system delivers cosmetic-enhancing substances into the body. In several embodiments, the delivery system delivers biomolecules, such as polynucleotides and proteins. It delivers substances, peptides, lipids, small molecules, or combinations thereof into the body. In several embodiments, the delivery system delivers polynucleotides for gene therapy. In some embodiments, the delivery system delivers therapeutic RNA. In terms of form, delivery systems deliver therapeutic proteins or peptides. In the application configuration, the delivery system delivers an antibody or a fragment thereof. In some embodiments, The delivery system delivers liposomes. In some embodiments, the delivery system is The system delivers anticancer drugs. In some embodiments, the delivery system delivers antimicrobial agents. In some embodiments, the delivery system delivers a wound healing agent. The polysaccharides are pullulan, hyaluronic acid, alginate, chitosan, dextran, cellulose This includes a delivery system or a combination thereof. In some embodiments, the delivery system is a drug delivery system. These are material capsules, such as PLANTCAPS® (Capsugel). In some embodiments, the delivery system is a skin filler, for example, JUVEDERM (registered It is a trademark (Allergan).

[0094] In some embodiments, the present disclosure provides for the purification of a poly(V) by the method provided herein. The present invention provides a pharmaceutical composition containing sugar. In some embodiments, the pharmaceutical composition is an antimicrobial agent. It contains polysaccharides with activity. For example, sulfated polysaccharides from seaweed have activity against plaque bacteria. Antimicrobial and antibiofilm activity, as well as antioxidants, anticoagulants, and immunostimulants. It may possess activity. For example, Barahona et al., Bioactive Ca rbohydrates and Dietary Fiber 4(2):125-1 38, 2014, and Xu et al., Marine Drugs 15(12) See 388,2017. In some embodiments, the polysaccharide includes chitosan. In further embodiments, from lactic acid bacteria, for example, Lactobacillus, Le uconostoc, Pediococcus, Lactococcus, Streptococcus ococcus, Aerococcus, Carnobacterium, Entero coccus, Oenococcus, Sporolactobacillus, Tet From the genera ragenococcus, Vagococcus, and Weissella Polysaccharides possess beneficial antitumor activity, anti-atherosclerotic effects, immunomodulatory activity, and prebiotic effects. It may be possible. For example, Lynch et al., Ann Rev Food Sci T See echnol 9:155-176,2018. In some embodiments, The pharmaceutical composition contains polysaccharides from seaweed. In some embodiments, the pharmaceutical composition is Contains polysaccharides derived from lactic acid bacteria.

[0095] In some embodiments, the present disclosure provides for the purification of a poly(V) by the method provided herein. Provides sugar. In some embodiments, polysaccharides are used in the vaccines described herein. It is used as such. In some embodiments, polysaccharides are used as vaccine adjuvants. In some embodiments, the polysaccharide is a protein or polypeptide as described herein. The cytoplasm, for example, is further conjugated to a protein or polypeptide vaccine antigen. In some embodiments, polysaccharides are used in the drug delivery systems described herein. It is used. In some embodiments, the polysaccharide is used in the pharmaceutical composition described herein. It is used.

[0096] All references cited herein, including patents, patent applications, papers, textbooks, etc. and the references cited therein (to the extent that they have not yet been cited) The entire text is incorporated herein by reference. [Examples]

[0097] Example 1. Test two different serotypes (serotypes 3 and 4) of Streptococcus using the following procedure. Polysaccharides were isolated from occus cells.

[0098] 1) Precipitation of DNA using CTAB and NaCl,

[0099] 2) Deep filtration to remove sediment,

[0100] 3) Precipitation with KI to remove CTAB,

[0101] 4) Centrifugation to remove precipitate,

[0102] 5) Carbon filtration to remove proteins and other impurities.

[0103] 6) SARTOBIND phenyl membrane chromatography for further removal of impurities Ceramic hydroxyapatite type (CHT) column chromatography bonded to Yee, 7) Tangential flow filtration for polysaccharide concentration.

[0104] Reference cells were subjected to a solution containing CTAB but without NaCl. Test cells were subjected to CTA The sample was subjected to a solution containing B and 400 mM NaCl.

[0105] The overall polysaccharide yields are shown in Table 1.

[0106] TIFF2026048886000001.tif31170

[0107] As shown in Table 1, cells that came into contact with a solution containing NaCl showed signs of Strepto A 9% increase in polysaccharide yield in coccus serotype 3 cells (from 43% to 52%), and A 17% increase in polysaccharide yield in Streptococcus serotype 4 cells (from 61%) It resulted in up to 78%.

[0108] Table 2 lists exemplary CTAB concentrations from Step 1. Further preferred salts and so The concentrations of these are listed in Table 3. TIFF2026048886000002.tif84170 TIFF2026048886000003.tif212170 TIFF2026048886000004.tif166170

[0109] Example 2. Use the following procedure to analyze polysaccharides, for example, positively charged, negatively charged, and / or neutral polysaccharides (P Luran (and other similar substances) are isolated from Aureobasidium pullulans cells. The cells are A. pullulans var. pullulans, A. pullulans It is either s var. melanogenicum or both. The cells are A. pullu lans MC571 shares, MC573 shares, MC574 shares, MC576 shares, MC711 shares, MC737, MC745, MC767, CBS701.76, and CBS10 Contains one or more of the 5.22 strains.

[0110] 1) DNA precipitation using CTAB and salts,

[0111] 2) Deep filtration to remove sediment,

[0112] 3) Precipitation with KI to remove CTAB,

[0113] 4) Centrifugation to remove precipitate,

[0114] 5) Carbon filtration to remove proteins and other impurities.

[0115] 6) SARTOBIND phenyl membrane chromatography for further removal of impurities Ceramic hydroxyapatite type (CHT) column chromatography bonded to Yee,

[0116] 7) Tangential flow filtration for polysaccharide concentration.

[0117] Table 2 provides the concentrations of CTAB used in the procedure. The salts used in the procedure... The concentrations and types are provided in Table 3.

[0118] Example 3. Using the procedure described in Example 2, polysaccharides, for example, positively charged, negatively charged, and / or Neutral polysaccharides are isolated from Staphylococcus aureus cells. The cells are Contains one or more of the following S. aureus serotypes: 1, 2, 5, 8, 336, or NT. The concentrations of CTAB used in the procedure are provided in Table 2. Table 3 provides the concentrations and types of salts used.

[0119] Example 4. Using the procedure described in Example 2, polysaccharides, for example, positively charged, negatively charged, and / or Neutral polysaccharides are isolated from Salmonella enterica cells. The cells are S .enterica serotypes Typhi, Typhimurium, Choleraesu is, Schwarzengrund, Derby, Haifa, Stanley, Ne wport, Virchow, Paratyphi, Singapore, Agona, Panama, Blockley, Anatum, Infantis, Enteriti dis, Heidelberg, Muenchen, Javiana, Montevid Contains one or more of eo, Thompson, or Oranienburg. Table 2 provides the concentrations of CTAB used in the procedure. The concentrations and types are provided in Table 3.

[0120] Example 5. Using the procedure described in Example 2, polysaccharides, for example, positively charged, negatively charged, and / or Neutral polysaccharides are isolated from Neisseria meningitidis cells. This refers to N. meningitidis serotype A, B, C, W, or Y (one or more). Contains. The concentrations of CTAB used in the procedure are provided in Table 2. The concentrations and types of salts used are provided in Table 3.

[0121] Example 6. Using the procedure described in Example 2, polysaccharides, for example, positively charged, negatively charged, and / or This involves isolating neutral polysaccharides from Mycobacterium tuberculosis cells. The cells are one of the following M. tuberculosis serotypes: 4, 8, 9, or NT. The above is included. The concentrations of CTAB used in the procedure are provided in Table 2. Table 3 provides the concentrations and types of salts used.

[0122] Example 7. Using the procedure described in Example 2, polysaccharides, for example, positively charged, negatively charged, and / or The neutral polysaccharide is isolated from Aspergillus fumigatus cells. Table 2 provides the concentrations of CTAB used in the procedure. Table 3 shows the different call types.

[0123] Example 8. Using the procedure described in Example 2, polysaccharides, for example, positively charged, negatively charged, and / or Neutral polysaccharides are isolated from Candida albicans cells. The cells are C. al Contains one or more of bicans serotype A or B. C used in the procedure The concentrations of TAB are provided in Table 2. The concentrations and types of salts used in the procedure are shown in Table 3. provide.

[0124] Example 9. Using the procedure described in Example 2, polysaccharides, for example, positively charged, negatively charged, and / or Neutral polysaccharides are isolated from Cryptococcus neoformans cells. The cells contain one of the following C. neoformans serotypes: A, D, or AD hybrid. It contains the above. The concentrations of CTAB used in the procedure are provided in Table 2. The concentrations and types of salts used are provided in Table 3.

[0125] Example 10. Using the procedure described in Example 2, polysaccharides, for example, positively charged, negatively charged, and / or Neutral polysaccharides are isolated from Histoplasma capsulatum cells. It contains one or more of H. capsulatum serotypes 1, 2, 3, 4, or 5. The concentrations of CTAB used in the procedure are provided in Table 2. Table 3 provides the concentrations and types of salts used.

Claims

1. A method for purifying polysaccharides from cell lysates, wherein the method is a. The cell lysate containing polysaccharides and impurities is partially purified to obtain a clarified crude lysate. To obtain things, b. The clarified crude solution is mixed with a neutralization solution containing approximately 100 mM to approximately 2 M of salt. This involves forming a neutralized solution, c. The neutralized solution is mixed with cetyltrimethic acid bromide at a concentration of approximately 0.1% v / v to approximately 6% v / v. Mix with a precipitate solution containing ammonium (CTAB) to obtain the first supernatant and the first precipitate. To form, d. Separating the first precipitate from the first supernatant, A method wherein the polysaccharide is substantially located in the first supernatant.

2. The cell lysates are partially purified by precipitation, centrifugation, filtration, or similar methods. The method according to claim 1, including a combination of the above.

3. The filtration method may be deep filtration, tangential flow filtration (TFF), sterile filtration, or a combination thereof. The method according to claim 2, including the method described in claim 2.

4. The neutralization solution contains approximately 200 mM to approximately 1 M of salt, as described in any one of claims 1 to 3. Method of loading.

5. The method according to claim 4, wherein the neutralization solution contains a salt concentration of approximately 250 mM to approximately 650 mM.

6. The method according to claim 5, wherein the neutralization solution contains a salt concentration of approximately 300 mM to approximately 500 mM.

7. | ４ 、、!| ４ ||、|||| ２ 、||| ２ 、! a ３ PO ４ The method according to claim 6, or a combination thereof.

8. The neutralization solution contains approximately 440 mM NaCl, as described in any one of claims 1 to 7. The method.

9. (b) The clarified crude solution in (b) is mixed with the neutralizing solution, and filtration Performed simultaneously with or before the step, and the filtration step is before (c), claim The method described in any one of items 1 to 8.

10. The mixing in (b) is performed simultaneously with the filtration step, according to claim 9. Method of description.

11. The method according to claim 9 or 10, wherein the filtration includes tangential flow filtration (TFF).

12. Claims 1 to 11, wherein the precipitated solution contains about 0.2% v / v to about 5% v / v of CTAB. The method described in any one of the items.

13. The precipitated solution contains approximately 0.5% v / v to approximately 4% v / v of CTAB, as described in claim 12. Method of loading.

14. The precipitated solution contains about 1% v / v to about 3% v / v of CTAB, as described in claim 13. method.

15. The cell lysate contains positively charged polysaccharides, neutral polysaccharides, negatively charged polysaccharides, or combinations thereof. The method according to any one of claims 1 to 14.

16. The positively charged polysaccharide, the neutral polysaccharide, the negatively charged polysaccharide, or a combination thereof is substantially The method according to any one of claims 1 to 15, wherein the first supernatant is contained in the first supernatant.

17. The impurity is substantially present in the first precipitate, according to any one of claims 1 to 16. Method of description.

18. The method according to any one of claims 1 to 17, wherein the impurity comprises a polynucleotide. 。

19. Claim that the polynucleotide is DNA, RNA, or a combination thereof. The method described in 18.

20. (b), (c), or both of the mixing is done by shaking, stirring, or pumping. The method according to any one of claims 1 to 19, performed by ng.

21. The separation is carried out by centrifugation, filtration, or a combination thereof. The method according to any one of claims 1 to 20.

22. The first supernatant is subjected to ultrafiltration, diafiltration, or a combination thereof, and the retaining liquid The process further comprises generating a permeate, wherein the polysaccharide is substantially present in the retaining liquid, The CTAB is substantially present in the permeate, as described in any one of claims 1 to 21. Law.

23. The aforementioned ultrafiltration, dialysfiltration, or a combination thereof is tangential flow filtration (TFF). The method according to claim 22.

24. The method according to claim 23 or 24, further comprising subjecting the retaining liquid to carbon filtration. 。

25. The method according to claim 24, further comprising subjecting the retaining solution to chromatography. 。

26. Potassium iodide (KI) is added to the first supernatant to obtain a second precipitate and a second supernatant. The further comprising forming the polysaccharide substantially present in the second supernatant, and the CT The method according to any one of claims 1 to 21, wherein AB is substantially in the second precipitate. Law.

27. The method described above does not include a chromatography step before the addition of KI, as per claim 1 to The method described in any one of paragraphs 26.

28. The second supernatant is separated from the second precipitate by centrifugation, filtration, or a combination thereof. The method according to claim 27, further comprising separating.

29. The method according to claim 28, further comprising subjecting the second supernatant to carbon filtration.

30. The claim 29 further comprises subjecting the second supernatant to chromatography. method.

31. The above chromatography is performed on ceramic hydroxyapatite type (CHT) chromatography. Mathography, hydrophobic interaction chromatography (HIC), or a combination thereof The method according to claim 25 or 30, including the addition of a fermented rice bran.

32. Claim 3, wherein the chromatography includes CHT chromatography and HIC chromatography. The method described in 1.

33. The HIC is performed using a chromatography column or membrane containing phenyl groups. The method according to claim 32.

34. A method according to claim 33, after chromatography, for concentrating the polysaccharide, limited External filtration, diafiltration, or a combination thereof.

35. Claim 3, wherein the ultrafiltration, dialysfiltration, or combination thereof is tangential flow filtration. The method described in 4.

36. The above method involves a first precipitate containing more than 10% of the polysaccharide in the cell lysate during the purification process. The method according to any one of claims 1 to 35, wherein no urinal material is included.

37. The positively charged polysaccharide, the neutral polysaccharide, the negatively charged polysaccharide, or a combination thereof is substantially The method according to any one of claims 1 to 36, wherein precipitation does not occur during the purification process.

38. The positively charged polysaccharide, the neutral polysaccharide, the negatively charged polysaccharide, or a combination thereof, The method according to any one of claims 1 to 37, which is not isolated from.

39. More than 90% (v / v) of negatively charged polysaccharides are present in the first supernatant, and less than 10% (v / v). The negatively charged polysaccharide is present in the first precipitate, according to any one of claims 1 to 38. Law.

40. More than 98% (v / v) of negatively charged polysaccharides are present in the first supernatant, and less than 2% (v / v) The method according to claim 39, wherein a negatively charged polysaccharide is present in the first precipitate.

41. The claim states that the yield percentage of the polysaccharide is greater than 30% of the polysaccharide in the cell lysate. The method described in any one of items 1 to 40.

42. The yield percentage of the polysaccharide is greater than 40% of the polysaccharide in the cell lysate. The method according to claim 41.

43. The yield percentage of the polysaccharide is greater than 50% of the polysaccharide in the cell lysate. The method according to claim 42.

44. The yield percentage of the polysaccharide is greater than 60% of the polysaccharide in the cell lysate. The method according to claim 43.

45. The yield percentage of the polysaccharide is 35% to 80% relative to the polysaccharide in the cell lysate. The method according to claim 44.

46. The cell lysate is derived from bacterial cells or fungal cells, any one of claims 1 to 45. The method described in section [section number].

47. The aforementioned cells are Staphylococcus cells, Streptococcus cells, Salmonella cells, Neisseria cells, Mycobacterium cells The method according to claim 46, wherein the cells are Aureobasidium cells.

48. Claim 4, wherein the cells are Aureobasidium pullulans cells. The method described in 7.

49. The method according to claim 47, wherein the cells are Streptococcus cells.

50. The aforementioned Streptococcus cells are serotypes 1, 2, 3, 4, 5, 6A, 6B, 7 F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C The claim 49 is characterized by being 19A, 19F, 20A, 22F, 23F, or 33F. method.

51. The Streptococcus cells are serotype 3 or 4, as described in claim 50. The method.

52. A method for purifying polysaccharides from cell lysates, wherein the method is a. The cell lysate containing impurities and polysaccharides is partially purified to obtain a clarified crude lysate. To obtain things, b. The clarified crude solution contains approximately 400 mM sodium chloride (NaCl) Mixing with a neutralizing solution to form a neutralized solution, c. The neutralized solution is then mixed with cetyltrimethylammonium bromide at a concentration of approximately 1.0% v / v. (CTAB) is mixed to form a first supernatant and a first precipitate, d. Separating the first precipitate from the first supernatant, e. The first supernatant is mixed with potassium iodide (KI) to form the second precipitate and the second supernatant. Mixing with, f. Separating the second precipitate from the second supernatant, A method wherein the polysaccharide is substantially located in the second supernatant.

53. A method for producing a polysaccharide vaccine, wherein the method is described in any one of claims 1 to 52. A method comprising purifying a polysaccharide according to the method described to obtain the polysaccharide vaccine.

54. The aforementioned polysaccharides include chitosan, glucan, mannose, inulin, galactose, and galacturo. 2-acetamido-4-amino-2,4,6-trideoxygalactose (AAT) Galp), glucuronic acid, glucose, rhamnose, N-acetylmannosamine (Ma nNAc), N-acetyl-L-fucosamine (FucNAc), beta-galactofurano The method according to claim 53, comprising -(beta-Galf), or a combination thereof. 。

55. Claim 5, wherein the polysaccharide is further conjugated with a protein or polypeptide. The method described in 3 or 54.

56. A vaccine comprising a polysaccharide purified by the method according to any one of claims 1 to 52. 。

57. The aforementioned polysaccharides include chitosan, glucan, mannose, inulin, galactose, and galacturo. 2-acetamido-4-amino-2,4,6-trideoxygalactose (AAT) Galp), glucuronic acid, glucose, rhamnose, N-acetylmannosamine (Ma nNAc), N-acetyl-L-fucosamine (FucNAc), beta-galactofurano The frame according to claim 56, comprising a ce (beta-galf), or a combination thereof Ding.

58. A delivery cis comprising a polysaccharide purified by the method described in any one of claims 1 to 52. Tem.

59. The aforementioned polysaccharides include pullulan, hyaluronic acid, alginate, chitosan, dextran, and cellulite. The delivery system according to claim 58, comprising loin or a combination thereof.

60. A pharmaceutical compound comprising a polysaccharide purified by the method described in any one of claims 1 to 52. A finished product.

61. The pharmaceutical composition according to claim 60, wherein the polysaccharide comprises chitosan.

62. A polysaccharide purified by the method described in any one of claims 1 to 52.

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