Purification of saccharides

Abstract

The invention relates to purification of saccharides. The invention relates to a method for purifying bacterial polysaccharides, in particular for removing impurities from cell lysates of polysaccharide-producing bacteria, comprising: a) acid hydrolysis; b) a first ultrafiltration / diafiltration-(UFDF-1); b) carbon filtration; c) chromatography; and d) a second ultrafiltration / diafiltration-(UFDF-2).

Description

[0001] This application is a divisional application of the invention patent application with the application date of February 17, 2021, application number "202180021155.1", and name "Purification of Carbohydrates".

[0002] Sequence Listing References

[0003] The parent application of this divisional application was filed electronically via EFS-Web and contains an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing with the title "PC72592_ST25.txt" created on January 29, 2021 and a size of 34KB. The sequence listing contained in this .txt file is part of the specification and is incorporated herein by reference in its entirety. This divisional application submits a sequence listing in ST.26 format. Technical Field

[0004] The present invention relates to a method for purifying bacterial polysaccharides, in particular for removing impurities from cell lysates of polysaccharide-producing bacteria. Background Art

[0005] Bacterial polysaccharides, especially capsular polysaccharides, are important immunogens found on the surface of bacteria and are associated with a variety of bacterial diseases. This has led to them becoming an important component of vaccine design. They have proven useful for eliciting immune responses, especially when linked to carrier proteins.

[0006] Bacterial polysaccharides are typically produced by fermentation of bacteria such as Streptococci (e.g., S. pneumoniae, S. pyogenes, S. agalactiae, or Group C and G Streptococci), Staphylococci (e.g., Staphylococcus aureus), Haemophilus (e.g., Haemophilus influenzae), Neisseria (e.g., Neisseria meningitidis), Escherichia (e.g., Escherichia coli), and Klebsiella (e.g., Klebsiella pneumoniae).

[0007] Typically, bacterial polysaccharides are produced using batch, fed-batch or continuous cultures in complex media.

[0008] There is a need for a robust and efficient purification process that can be used for large-scale production of post-fermentation bacterial polysaccharides.

[0009] There is also a need for a simplified purification process to reduce the soluble protein content in bacterial lysates and eliminate the inefficiencies of current purification processes to produce substantially purified bacterial saccharides suitable for incorporation into vaccines. Summary of the invention

[0010] The present invention provides a method for purifying sugars from a solution comprising sugars derived from bacteria and post-fermentation contaminants, wherein the method comprises the following steps: (a) acid hydrolysis; (b) first ultrafiltration / diafiltration-(UFDF-1); (b) carbon filtration; (c) chromatography; and (d) second ultrafiltration / diafiltration-(UFDF-2). In one embodiment, the method further comprises a flocculation step after the acid hydrolysis of step (a).

[0011] In another embodiment of the above method, the bacterium is a gram-positive bacterium. In one aspect, the bacterium is any one of Streptococcus, Staphylococcus, Enterococcus, Bacillus, Corynebacterium, Listeria, Erysipelothrix or Clostridium. In another aspect, the bacterium is any one of Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Group C and Group G Streptococcus or Staphylococcus aureus.

[0012] In another embodiment of the above method, the bacterium is a gram-negative bacterium. In one aspect, the bacterium is any one of the genus Haemophilus, Neisseria, Escherichia or Klebsiella. In another aspect, the bacterium is Haemophilus influenzae, Neisseria meningitidis, Escherichia coli or Klebsiella pneumoniae. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Depicts the K of the O1V1 variant after release into culture medium p O-Ag (top) and purified K p SEC-HPLC chromatogram of O-Ag (bottom).

[0014] Figure 2 Depicts the K of the O1V2 variant after release into culture medium p O-Ag (top) and purified K p SEC-HPLC chromatogram of O-Ag (bottom).

[0015] Figure 3 Depicts the K of O2V1 variants after release in culture medium p O-Ag (top) and purified K p SEC-HPLC chromatogram of O-Ag (bottom).

[0016] Figure 4 Depicts the K of O2V2 variants after release in culture medium p O-Ag (top) and purified K p SEC-HPLC chromatogram of O-Ag (bottom). DETAILED DESCRIPTION

[0017] The present invention provides a method for purifying sugars derived from bacteria from a solution comprising sugars and post-fermentation contaminants, wherein the method comprises the following steps: (a) acid hydrolysis; (b) first ultrafiltration / diafiltration-(UFDF-1); (b) carbon filtration; (c) chromatography; and (d) second ultrafiltration / diafiltration-(UFDF-2).

[0018] In one embodiment, the method further comprises a flocculation step after the acid hydrolysis of step (a).

[0019] In another embodiment of the above method, the chromatography of step (c) comprises IEX membrane chromatography or hydrophobic interaction chromatography (HIC) or both.

[0020] In another embodiment of the above method, the bacterium is a gram-positive bacterium. In one aspect, the bacterium is any one of Streptococcus, Staphylococcus, Enterococcus, Bacillus, Corynebacterium, Listeria, Erysipelothrix or Clostridium. In another aspect, the bacterium is any one of Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Group C and Group G Streptococcus or Staphylococcus aureus.

[0021] In another embodiment of the above method, the bacterium is a gram-negative bacterium. In one aspect, the bacterium is any one of the genus Haemophilus, Neisseria, Escherichia or Klebsiella. In another aspect, the bacterium is Haemophilus influenzae, Neisseria meningitidis, Escherichia coli or Klebsiella pneumoniae.

[0022] On the other hand, the bacterium is Escherichia coli including a carbohydrate having a structure selected from any one of the following: formula O1, formula O1A, formula O1B, formula O1C, formula O2, formula O3, formula O4, formula O4:K52, formula O4:K6, formula O5, formula O5ab, formula O5ac, formula O6, formula O6:K2; K13; K15, formula O6:K54, formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18, formula O18A, formula O18ac, formula O18A1, formula O18B, formula O18B1, formula O19, formula O20, formula O21, formula O22, formula O23, formula O23A, formula O24, formula O25 O25, O25a, O25b, O26, O27, O28, O29, O30, O32, O33, O34, O35, O36, O37, O38, O39, O40, O41, O42, O43, O44, O45, O45rel, O45 O46、O48、O49、O50、O51、O52、O53、O54、O55、O56、O57、O58、O59、O60、O61、O62、O62D1、O63、O64、O65、O66、O68、O69、O70、O71、 Formula O73, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, O98, O99, O100, O101, O102, O103, O104, O105, O106, O107, O108, O109, O110, O111, O112, O113, O114, O115, O116, O117, O118, O119 119, O120, O121, O123, O124, O125, O126, O127, O128, O129, O130, O131, O132, O133, O134, O135, O136, O137, O138, O139, O140, O141、O142、O143、O144、O145、O146、O147、O148、O149、O150、O151、O152、O153、O154、O155、O156、O157、O158、O159、O160、O161、Formula O162, formula O163, formula O164, formula O165, formula O166, formula O167, formula O168, formula O169, formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186 or formula O187.

[0023] On the other hand, the bacterium is Klebsiella pneumoniae comprising a carbohydrate having a structure selected from any one of the following: Formula K.O1.1, Formula K.O1.2, Formula K.O1.3, Formula K.O1.4, Formula K.O2.1, Formula K.O2.2, Formula K.O2.3, Formula K.O2.4, Formula K.O3, Formula K.O4, Formula K.O5, Formula K.O7, Formula K.O12 or Formula K.O8.

[0024] 1. Purification of bacterial polysaccharides

[0025] 1.1 Starting materials

[0026] The method of the present invention can be used to purify a bacterial polysaccharide from a solution comprising said polysaccharide and contaminants.

[0027] Bacterial cells

[0028] The source of the bacterial polysaccharide to be purified according to the present invention is bacterial cells, in particular pathogenic bacteria.

[0029] Non-limiting examples of Gram-positive bacteria used according to the present invention are Streptococcus (e.g., Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, or Group C and Group G Streptococci), Staphylococcus (e.g., Staphylococcus aureus), Enterococcus, Bacillus, Corynebacterium, Listeria, Erysipelothrix, and Clostridium. Non-limiting examples of Gram-negative bacteria used according to the present invention include Haemophilus (e.g., Haemophilus influenzae), Neisseria (e.g., Neisseria meningitidis), Escherichia (e.g., Escherichia coli), and Klebsiella (e.g., Klebsiella pneumoniae).

[0030] In one embodiment, the source of bacterial polysaccharides used in accordance with the present invention is selected from the group consisting of: Aeromonas hydrophila and other species; Bacillus anthracis; Bacillus cereus; Clostridium species that produce Botulinum neurotoxin; Brucella abortus; Brucella melitensis; Brucella suis; Burkholderia mallei (formally known as Pseudomonas mallei); Burkholderia pseudomallei (formerly known as Pseudomonas pseudomallei); Campylobacter jejuni; Chlamydia psittaci; Chlamydia trachomatis; Clostridium botulinum; botulinum; Clostridium dificile; Clostridium perfringens; Coccidioides immitis; Coccidioides posadasii; Cowdria ruminantium (heartwater disease); Coxiella burnetii; Enterococcus faecalis; Enterovirulent Escherichia coli group (EEC group), such as enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli O157:H7 (EHEC), and enteroinvasive Escherichia coli (EIEC); Ehrlichia spp., such as Ehrlichia chaffeensis. chajfeensis; Francisella tularensis; Legionella pneumophilia; Liberobacter africanus; Liberobacter asiaticus; Listeria monocytogenes;Miscellaneous enterics, such as Klebsiella, Enterobacter, Proteus, Citrobacter, Aerobacter, Providencia, and Serratia; Mycobacterium bovis; Mycobacterium tuberculosis; Mycoplasma capricolum: Mycoplasma mycoides ssp mycoides; Peronosclerospora philippinensis; Phakopsora pachyrhizi; Plesiomonas shigelloides; Ralstonia solanacearum race 3 biotype 2 3, biovar 2); Rickettsia prowazekii; Rickettsia rickettsii; Salmonella spp.; Schlerophthora rayssiae var zeae; Shigella spp.; Staphylococcus aureus; Streptococcus; Synchytrium endobioticum; Vibrio cholerae non-01; Vibrio cholerae 01; Vibrio parahaemolyticus and other Vibrio species; Vibrio vulnificus; Xanthomonas oryzae; oryzae); Xylella fastidiosa (citrus variegated chlorosis strain); Yersinia enterocolitica and Yersinia pseudotuberculosis; and Yersinia pestis.

[0031] The polysaccharide to be purified may be associated with a cellular component such as the cell wall. Associated with the cell wall means that the polysaccharide is a component of the cell wall itself and / or is attached to the cell wall directly or indirectly through an intermediate molecule, or is a transient outer layer of the cell wall (e.g., certain bacterial strains exude capsular polysaccharides, also referred to in the art as 'exopolysaccharides').

[0032] In some embodiments, the polysaccharide extracted from the bacteria is a capsular polysaccharide, a subcapsular polysaccharide or a lipopolysaccharide. In a preferred embodiment, the polysaccharide is a capsular polysaccharide.

[0033] In one embodiment, the source of bacterial capsular polysaccharide is Escherichia coli. In another embodiment, the source of bacterial capsular polysaccharide is the Escherichia coli part of the diarrheagenic Escherichia coli group (EEC group), such as enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), O157:H7 enterohemorrhagic Escherichia coli (EHEC) or enteroinvasive Escherichia coli (EIEC). In one embodiment, the source of bacterial capsular polysaccharide is uropathogenic Escherichia coli (UPEC).

[0034] In another embodiment, the source of the bacterial capsular polysaccharide is an E. coli serotype selected from the group consisting of serotypes O157:H7, O26:H11, O111:H- and O103:H2. In one embodiment, the source of the bacterial capsular polysaccharide is an E. coli serotype selected from the group consisting of serotypes O6:K2:H1 and O18:K1:H7. In one embodiment, the source of the bacterial capsular polysaccharide is an E. coli serotype selected from the group consisting of serotypes O45:K1, O17:K52:H18, O19:H34 and O7:K1. In one embodiment, the source of the bacterial capsular polysaccharide is an E. coli serotype O104:H4. In one embodiment, the source of the bacterial capsular polysaccharide is E. coli serotype O1:K12:H7. In one embodiment, the source of the bacterial capsular polysaccharide is E. coli serotype O127:H6. In one embodiment, the source of bacterial capsular polysaccharide is Escherichia coli serotype O139:H28. In one embodiment, the source of bacterial capsular polysaccharide is Escherichia coli serotype O128:H2.

[0035] In another embodiment, the source of the bacterial capsular polysaccharide is Neisseria meningitidis. In one embodiment, the source of the bacterial capsular polysaccharide is Neisseria meningitidis serogroup A (MenA), Neisseria meningitidis serogroup W135 (MenW135), Neisseria meningitidis serogroup Y (MenY), Neisseria meningitidis serogroup X (MenX) or Neisseria meningitidis serogroup C (MenC). In one embodiment, the source of the bacterial capsular polysaccharide is Neisseria meningitidis serogroup A (MenA). In one embodiment, the source of the bacterial capsular polysaccharide is Neisseria meningitidis serogroup W135 (MenW135). In one embodiment, the source of the bacterial capsular polysaccharide is Neisseria meningitidis serogroup Y (MenY). In one embodiment, the source of the bacterial capsular polysaccharide is Neisseria meningitidis serogroup C (MenC). In one embodiment, the source of the bacterial capsular polysaccharide is Neisseria meningitidis serogroup X (MenX).

[0036] In another embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae. In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O1 (O1), Klebsiella pneumoniae serogroup O2 (O2), Klebsiella pneumoniae serogroup O2ac (O2ac), Klebsiella pneumoniae serogroup O3 (O3), Klebsiella pneumoniae serogroup O4 (O4), Klebsiella pneumoniae serogroup O5 (O5), Klebsiella pneumoniae serogroup O7 (O7), Klebsiella pneumoniae serogroup O8 (O8) or Klebsiella pneumoniae serogroup O9 (O9). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O1 (O1). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O2 (O2). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O2ac (O2ac). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O3 (O3). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O4 (O4). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O5 (O5). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O7 (O7). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O8 (O8). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O9 (O9).

[0037] 1.1.2. Bacterial cell growth

[0038] Typically, the polysaccharide is produced by culturing the bacteria in a medium (eg, solid or preferably liquid medium). The polysaccharide is then prepared by treating the bacterial cells.

[0039] Therefore, in one embodiment, the starting material of the method of the present invention is a bacterial culture, and preferably a liquid bacterial culture (eg, a fermentation broth).

[0040] Bacterial cultures are usually obtained by batch culture, fed batch culture or continuous culture (see, for example, WO 2007 / 052168 or WO 2009 / 081276). During continuous culture, fresh culture medium is added to the culture at a fixed rate, and cells and culture medium are removed at a rate to maintain a constant culture volume.

[0041] Populations of organisms are typically expanded from seed vial to seed vial and passaged through one or more seed fermentors of increasing volume until a production-scale fermentation volume is reached.

[0042] 1.1.3 Pretreatment of bacterial cells to obtain starting materials

[0043] Typically, small amounts of polysaccharides are released into the culture medium during bacterial growth, so the starting material may be the supernatant from a centrifuged bacterial culture. Typically, however, the starting material will be prepared by treating the bacteria themselves, thereby releasing the polysaccharides.

[0044] Optionally, after cell growth, the bacterial cells are inactivated. This is particularly true when using pathogenic bacteria. A suitable inactivation method is, for example, treatment with phenol:ethanol, such as described in Fattom et al. (1990) Infect Immun. 58(7): 2367-74. In the following examples, the bacterial cells may or may not be inactivated in advance.

[0045] Polysaccharides can be released from bacteria by various methods including chemical, physical or enzymatic treatment (see for example WO2010151544, WO 2011 / 051917 or WO2007084856).

[0046] In one embodiment, the bacterial cells (inactivated or not) are treated in suspension in their original culture medium. Thus, the method can start with cells suspended in their original culture medium.

[0047] In another embodiment, the bacterial cells are centrifuged before releasing the capsular polysaccharide. Thus, the method can start with cells in the form of a wet cell paste. Alternatively, the cells are processed in a dry form. However, typically, after centrifugation, the bacterial cells are resuspended in an aqueous medium suitable for the next step of the method, such as in a buffer or distilled water. Prior to resuspension, the cells can be washed with this medium.

[0048] In one embodiment, the bacterial cells (e.g., in the form of a suspension in their original culture medium, in the form of a wet cell paste, in a dry form, or resuspended in an aqueous culture medium after centrifugation) are treated with a lytic agent. A "lytic agent" is any reagent that helps to decompose the cell wall. In one embodiment, the lytic agent is a detergent. As used herein, the term "detergent" refers to any anionic or cationic detergent that can cause bacterial cell lysis. Representative examples of such detergents for the inventive method include sodium deoxycholate (DOC), N-lauryl sarcosine (NLS), sodium chenodeoxycholate, and saponin (see WO2008 / 118752, page 13, line 14 to page 14, line 10). In one embodiment of the invention, the lytic agent for lysing bacterial cells is DOC.

[0049] In one embodiment, the dissolving agent is a non-animal derived dissolving agent. In one embodiment, the non-animal derived dissolving agent is selected from the group consisting of: decane sulfonic acid, tert-octylphenoxy 5-poly(ethylene oxide) ethanol (e.g., IGEPAL CA-630, CAS No.: 9002-93-1, available from Sigma Aldrich, St. Louis, MO), octylphenol ethylene oxide condensate (e.g., TRITON X-100, available from Sigma Aldrich, St. Louis, MO), sodium N-lauryl sarcosinate (NLS), lauryl iminodipropionate, sodium dodecyl sulfate, chenodeoxycholate, hyodeoxycholate, glycodeoxycholate, taurodeoxycholate, taurochenodeoxycholate, and cholate. In one embodiment, the non-animal derived dissolving agent is NLS.

[0050] In one embodiment, the bacterial cells (e.g., as a suspension in their original culture medium, as a wet cell paste, in a dried form, or resuspended in an aqueous culture medium after centrifugation) are treated with an enzyme to release the polysaccharide. In one embodiment, the bacterial cells are treated with an enzyme selected from the group consisting of lysostaphin, mutanolysin β-N-acetylglucosaminidase, and a combination of mutanolysin and β-N-acetylglucosaminidase. These act on bacterial peptidoglycans to release capsular saccharides for use in the present invention, but also result in the release of group-specific carbohydrate antigens. In one embodiment, the bacterial cells are treated with type II phosphodiesterase (PDE2).

[0051] Optionally, after the polysaccharide is released, the enzyme is inactivated. Suitable inactivation methods are, for example, heat treatment or acid treatment.

[0052] In one example, bacterial cells (eg, as a suspension in their original culture medium, as a wet cell paste, in a dried form, or resuspended in an aqueous culture medium after centrifugation) are subjected to high pressure treatment to release polysaccharides.

[0053] In another embodiment, the bacterial cells (e.g., in suspension in their original culture medium, in a wet cell paste, in a dried form, or resuspended in an aqueous culture medium after centrifugation) are chemically treated to release polysaccharides. In such embodiments, the chemical treatment may be, for example, hydrolysis using an alkali or acid (see, e.g., WO2007084856).

[0054] In one embodiment, the chemical treatment of the bacterial cells is an alkaline extraction (e.g., using sodium hydroxide). Alkaline extraction can cleave the phosphodiester bonds between the capsular sugars and the peptidoglycan backbone. In one embodiment, the base is selected from the group consisting of: NaOH, KOH, LiOH, NaHCO3, Na2CO3, KzCO3, KCN, Et3N, NH3, HzN2H2, NaH, NaOMe, NaOEt, and KOtBu. After the alkaline treatment, the reaction mixture can be neutralized. This can be achieved by adding an acid. In one embodiment, after the alkaline treatment, the reaction mixture is neutralized by an acid selected from the group consisting of: HCl, H 3 PO 4 , citric acid, acetic acid, nitrous acid and sulfuric acid.

[0055] In one embodiment, the bacterial cell chemical treatment is an acid treatment (e.g., sulfuric acid). In one embodiment, the acid is selected from the group consisting of: HCl, H 3 PO 4 , citric acid, acetic acid, nitrous acid and sulfuric acid. After the acid treatment, the reaction mixture can be neutralized. This can be achieved by adding a base. In one embodiment, after the acid treatment, the reaction mixture is neutralized by a base selected from the group consisting of: NaOH, KOH, LiOH, NaHCO3, Na2CO3, KzCO3, KCN, Et3N, NH3, HzN2H2, NaH, NaOMe, NaOEt and KOtBu.

[0056] 1.2 Flocculation

[0057] The method of the present invention comprises a flocculation step. The inventors have found that the process is rapid and simple and produces purified polysaccharides with low contamination.

[0058] Therefore, in the method of the present invention, the solution obtained by any of the methods described in section 1.1 above is treated by flocculation.

[0059] In the present invention, the term "flocculation" refers to a process in which colloids are separated from a suspension in the form of flocs or flakes due to the addition of a flocculant.

[0060] The flocculation step includes adding a "flocculant" to a solution including bacterial polysaccharides and contaminants. In one embodiment, the contaminants include bacterial cell debris, bacterial cell proteins, and nucleic acids. In one embodiment, the contaminants include bacterial cell proteins and nucleic acids.

[0061] As will be further disclosed below, the flocculation step may also comprise adjusting the pH before or after the addition of the flocculant. In particular, the solution may be acidified.

[0062] Furthermore, the addition of the flocculant and / or the adjustment of the pH may be performed at a temperature adjusted to a desired level.

[0063] The following steps can be performed in any order:

[0064] - Addition of flocculant followed by pH adjustment followed by temperature adjustment, or;

[0065] - Addition of flocculant followed by temperature adjustment followed by pH adjustment, or;

[0066] - pH adjustment followed by addition of flocculant followed by temperature adjustment, or;

[0067] - pH adjustment followed by temperature adjustment followed by addition of flocculant, or;

[0068] - adjusting the temperature, followed by addition of a flocculant, followed by adjustment of the pH, or;

[0069] - Adjust the temperature, then adjust the pH, then add the flocculant.

[0070] Additionally, after addition of the flocculant and / or adjustment of the pH, the solution may be held for a period of time to allow for the precipitation of flocs prior to downstream processing.

[0071] In the present invention, "flocculant" refers to an agent capable of allowing or promoting flocculation in a solution comprising a polysaccharide of interest and a pollutant by causing colloids and other suspended particles to aggregate in the form of flocs or flakes, while the polysaccharide of interest remains in solution.

[0072] In one embodiment of the present invention, the flocculant comprises a multivalent cation. In one embodiment, the flocculant is a multivalent cation. In a preferred embodiment, the multivalent cation is selected from the group consisting of aluminum, iron, calcium and magnesium. In one embodiment, the flocculant is a mixture of at least two multivalent cations selected from the group consisting of aluminum, iron, calcium and magnesium. In one embodiment, the flocculant is a mixture of at least three multivalent cations selected from the group consisting of aluminum, iron, calcium and magnesium. In one embodiment, the flocculant is a mixture of four multivalent cations consisting of aluminum, iron, calcium and magnesium.

[0073] In one embodiment, the flocculant comprises an agent selected from the group consisting of: alum (e.g., potassium alum, sodium alum, or ammonium alum), aluminum chloride hydrate, aluminum sulfate, calcium oxide, calcium hydroxide, iron (II) sulfate (ferrous sulfate), iron (III) chloride (ferric chloride), polyacrylamide, modified polyacrylamide, polyDADMAC, polyethyleneimine (PEI), sodium aluminate, and sodium silicate. In one embodiment, the flocculant is selected from the group consisting of: alum (e.g., potassium alum, sodium alum, or ammonium alum), aluminum chloride hydrate, aluminum sulfate, calcium oxide, calcium hydroxide, iron (II) sulfate (ferrous sulfate), iron (III) chloride (ferric chloride), polyacrylamide, modified polyacrylamide, polyDADMAC, sodium aluminate, and sodium silicate. In one embodiment, the flocculant is polyethyleneimine (PEI). In one embodiment, the flocculant comprises alum. In one embodiment, the flocculant is alum. In one embodiment, the flocculant comprises potassium alum. In one embodiment, the flocculant is potassium alum. In one embodiment, the flocculant comprises sodium alum. In one embodiment, the flocculant is sodium alum. In one embodiment, the flocculant comprises ammonium alum. In one embodiment, the flocculant is ammonium alum.

[0074] In one embodiment, the flocculant is a mixture of agents (e.g., two, three, or four agents) selected from the group consisting of: alum (e.g., potassium alum, sodium alum, or ammonium alum), aluminum chloride hydrate, aluminum sulfate, calcium oxide, calcium hydroxide, iron (II) sulfate (ferrous sulfate), iron (III) chloride (ferric chloride), polyacrylamide, modified polyacrylamide, polyDADMAC, polyethyleneimine (PEI), sodium aluminate, and sodium silicate. In one embodiment, the flocculant is selected from the group consisting of: alum (e.g., potassium alum, sodium alum, or ammonium alum), aluminum chloride hydrate, aluminum sulfate, calcium oxide, calcium hydroxide, iron (II) sulfate (ferrous sulfate), iron (III) chloride (ferric chloride), polyacrylamide, modified polyacrylamide, polyDADMAC, sodium aluminate, and sodium silicate.

[0075] In one embodiment, the flocculant is a mixture of two agents selected from the group consisting of: alum (e.g., potassium alum, sodium alum, or ammonium alum), aluminum chloride hydrate, aluminum sulfate, calcium oxide, calcium hydroxide, iron (II) sulfate (ferrous sulfate), iron (III) chloride (ferric chloride), polyacrylamide, modified polyacrylamide, polyDADMAC, sodium aluminate, and sodium silicate. In one embodiment, the flocculant is a mixture of at least three agents selected from the group consisting of: alum (e.g., potassium alum, sodium alum, or ammonium alum), aluminum chloride hydrate, aluminum sulfate, calcium oxide, calcium hydroxide, iron (II) sulfate (ferrous sulfate), iron (III) chloride (ferric chloride), polyacrylamide, modified polyacrylamide, polyDADMAC, sodium aluminate, and sodium silicate.

[0076] In one embodiment, the flocculant comprises an agent selected from the group consisting of: chitosan, mica, Moringa seeds (horseradish tree), gelatin, Aristolochia seeds (Nirmali nut tree), guar gum, and alginates (e.g., brown seaweed extracts). In one embodiment, the flocculant is selected from the group consisting of: chitosan, mica, Moringa seeds (horseradish tree), gelatin, Aristolochia seeds (Nirmali nut tree), guar gum, and alginates (e.g., brown seaweed extracts).

[0077] The concentration of the flocculant may depend on the reagent used, the polysaccharide of interest and the parameters of the flocculation step (eg temperature).

[0078] In embodiments where the flocculant comprises or is alum, a flocculant concentration between about 0.1% and 20% (w / v) may be used. Preferably, a flocculant concentration between about 0.5% and 10% (w / v) is used. Even more preferably, a flocculant concentration between about 1% and 5% (w / v) is used. Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0079] In one embodiment, a flocculant concentration of about 0.1% (w / v), about 0.25% (w / v), about 0.5% (w / v), about 1.0% (w / v), about 1.5% (w / v), about 2.0% (w / v), about 2.5% (w / v), about 3.0% (w / v), about 3.5% (w / v), about 4.0% (w / v), about 4.5% (w / v), about 5.0% (w / v), about 5.5% (w / v), about 6.0% (w / v), about 6.5% (w / v), about 7.0% (w / v), about 7.5% (w / v), about 8.0% (w / v), about 8.5% (w / v), about 9.0% (w / v), about 9.5% (w / v) or about 10% (w / v) is used. In one embodiment, a flocculant concentration of about 10.5% (w / v), about 11.0% (w / v), about 11.5% (w / v), about 12.0% (w / v), about 12.5% ​​(w / v), about 13.0% (w / v), about 13.5% (w / v), about 14.0% (w / v), about 14.5% (w / v), about 15.0% (w / v), about 15.5% (w / v), about 16.0% (w / v), about 16.5% (w / v), about 17.0% (w / v), about 17.5% (w / v), about 18.0% (w / v), about 18.5% (w / v), about 19.0% (w / v), about 19.5% (w / v) or about 20.0% (w / v) is used. In one embodiment, a flocculant concentration of about 0.5% (w / v), about 1.0% (w / v), about 1.5% (w / v), about 2.0% (w / v), about 2.5% (w / v), about 3.0% (w / v), about 3.5% (w / v), about 4.0% (w / v), about 4.5% (w / v), or about 5.0% (w / v) is used. In one embodiment, a flocculant concentration of about 1.0% (w / v), about 1.5% (w / v), about 2.0% (w / v), about 2.5% (w / v), about 3.0% (w / v), about 3.5% (w / v), or about 4.0% (w / v) is used.

[0080] In some embodiments of the invention, the flocculant is added over a period of time. In some embodiments of the invention, the flocculant is added over a period of time between a few seconds (e.g., 1 to 10 seconds) and about a month. In some embodiments, the flocculant is added over a period of time between about 2 seconds and about two weeks. In some embodiments of the invention, the flocculant is added over a period of time between about 1 minute and about a week. In some embodiments, the flocculant is added over a period of time between about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 130 minutes, about 140 minutes, about 150 minutes, about 160 minutes, about 170 minutes, about 180 minutes, about 190 minutes, about 200 minutes, about 210 minutes, about 220 minutes, about 230 minutes, about 240 minutes, about 250 minutes, about 260 minutes, about 270 minutes, about 280 minutes, about 290 minutes, about 300 minutes, about 310 minutes, about 320 minutes, about 330 minutes, about 340 minutes, about 350 minutes, about 360 minutes, about 370 minutes, about 380 minutes, about 390 minutes, about 400 minutes, about 410 minutes, about 420 minutes, about 430 minutes, about 440 minutes, about 450 minutes, about The flocculant is added within a time period of between 50 minutes, about 160 minutes, about 170 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours and about two days.

[0081] Thus, in certain embodiments, the flocculant is added over a period of between about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 130 minutes, about 140 minutes, about 150 minutes, about 160 minutes, about 170 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours, and about one day.

[0082] Preferably, the flocculant is added over a period of between about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 130 minutes, about 140 minutes, about 150 minutes, about 160 minutes, about 170 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours or about 12 hours and about one day.

[0083] In certain embodiments, the flocculant is added over a period of time between about 15 minutes and about 3 hours. In certain embodiments, the flocculant is added over a period of time between about 30 minutes and about 120 minutes.

[0084] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0085] In one embodiment, the amount of time for the reaction may be about 2 seconds, about 10 seconds, about 30 seconds, about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes, about 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes, about 145 minutes, about 150 minutes, about 155 minutes, about 160 minutes, about 170 minutes, about 3 hours, about 3.5 hours, or about 4 hours. The flocculant may be added within a period of about 1 hour, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days or about 15 days.

[0086] In one embodiment, the flocculant is added without stirring. In another embodiment, the flocculant is added with stirring. In another embodiment, the flocculant is added with mild stirring. In another embodiment, the flocculant is added with vigorous stirring.

[0087] The inventors further unexpectedly noted that flocculation was improved when performed at an acidic pH.

[0088] Thus, in one embodiment of the invention, the flocculation step is performed at a pH of less than 7.0, 6.0, 5.0 or 4.0. In a specific embodiment of the invention, the flocculation step is performed at a pH between 7.0 and 1.0. In one embodiment, the flocculation step is performed at a pH between 5.5 and 2.5, 5.0 and 2.5, 4.5 and 2.5, 4.0 and 2.5, 5.5 and 3.0, 5.0 and 3.0, 4.5 and 3.0, 4.0 and 3.0, 5.5 and 3.5, 5.0 and 3.5, 4.5 and 3.5 or 4.0 and 3.5. In one embodiment, the flocculation step is performed at a pH of about 5.5, about 5.0, about 4.5, about 4.0, about 3.5, about 3.0, about 2.5, about 2.0, about 1.5 or about 1.0. In one embodiment, the flocculation step is performed at a pH of about 4.0, about 3.5, about 3.0, or about 2.5. In one embodiment, the flocculation step is performed at a pH of about 3.5. Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0089] In one embodiment, the acidic pH is obtained by acidifying the solution obtained by any of the methods in Section 1.1 above or further clarified as disclosed in Section 1.2 with an acid. In one embodiment, the acid is selected from the group consisting of: HCl, H 3 PO 4 In one embodiment, the acid is an amino acid. In one embodiment, the acid is an amino acid selected from the group consisting of glycine, alanine, and glutamic acid. In one embodiment, the acid is HCl (hydrochloric acid). In one embodiment, the acid is sulfuric acid.

[0090] In one embodiment, the acid is added without stirring. Preferably, the acid is added with stirring. In one embodiment, the acid is added with mild stirring. In one embodiment, the acid is added with vigorous stirring.

[0091] In some embodiments of the invention, after addition of the flocculant (and optional acidification), the solution is allowed to stand for a period of time to allow the flocs to settle prior to downstream processing.

[0092] In some embodiments of the present invention, the flocculation step is carried out with a settling time between a few seconds (e.g., 2 to 10 seconds) to about 1 minute. Preferably, the settling time is at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 105, at least about 110, at least about 115, at least about 120, at least about 125, at least about 130, at least about 135, at least about 140, at least about 145, at least about 150, at least about 155, or at least about 160 minutes. Preferably, the settling time is less than one week, but the settling time can be longer.

[0093] Thus, in certain embodiments, the precipitation time is between about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 140, about 160, about 180, about 220, about 240, about 300, about 360, about 420, about 480, about 540, about 600, about 660, about 720, about 780, about 840, about 900, about 960, about 1020, about 1080, about 1140, about 1200, about 1260, about 1320, about 1380, about 1440 minutes, about two days, about three days, about four days, about five days, or about six days and 1 week.

[0094] In some embodiments of the present invention, the precipitation time is between a few seconds (e.g., 1 to 10 seconds) and about a month. In some embodiments, the precipitation time is between about 2 seconds and about two weeks. In some embodiments of the present invention, the precipitation time is between about 1 minute and about a week. In some embodiments, the precipitation time is between about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 130 minutes, about 140 minutes, about 150 minutes, about 160 minutes, about 170 minutes, about 180 minutes, about 190 minutes, about 200 minutes, about 210 minutes, about 220 minutes, about 230 minutes, about 240 minutes, about 250 minutes, about 260 minutes, about 270 minutes, about 280 minutes, about 290 minutes, about 300 minutes, about 310 minutes, about 320 minutes, about 330 minutes, about 340 minutes, about 350 minutes, about 360 minutes, about 370 minutes, about 380 minutes, about 390 minutes, about 400 minutes, about 410 minutes, about 420 minutes, about 430 minutes, about 440 minutes, about 450 minutes, about 460 minutes, about 470 minutes, about 480 minutes, about 490 minutes, about 500 minutes, about 510 minutes, about 510 minutes, about 520 minutes, about 530 minutes, 40 minutes, about 150 minutes, about 160 minutes, about 170 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours or about 24 hours and about two days.

[0095] Thus, in certain embodiments, the precipitation time is between about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 130 minutes, about 140 minutes, about 150 minutes, about 160 minutes, about 170 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours or about 12 hours and about one day.

[0096] Preferably, the precipitation time is between about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 130 minutes, about 140 minutes, about 150 minutes, about 160 minutes, about 170 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours or about 12 hours and about one day.

[0097] In certain embodiments, the precipitation time is between about 15 minutes and about 3 hours. In certain embodiments, the precipitation time is between about 30 minutes and about 120 minutes.

[0098] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0099] In certain embodiments, the precipitation time is about 2 seconds, about 10 seconds, about 30 seconds, about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes, about 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes, about 145 minutes, about 150 minutes, about 155 minutes, about 160 minutes, about 170 minutes, about 180 minutes, about 190 minutes, about 200 minutes, about 210 minutes, about 220 minutes, about 230 minutes, about 240 minutes, about 250 minutes, about 260 minutes, about 270 minutes, about 280 minutes, about 290 minutes, about 300 minutes, about 310 minutes, about 320 minutes, about 330 minutes, about 340 minutes, about 350 minutes, about 360 minutes, about 370 minutes, about 380 minutes, about 390 minutes, about 400 minutes, about 410 minutes, about 420 minutes, about 430 minutes, about 440 minutes about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days or about 15 days.

[0100] Preferably, the precipitation time is between about 5, about 10, about 15, about 20, about 25, about 30, about 60, about 90, about 120, about 180, about 220, about 240, about 300, about 360, about 420, about 480, about 540, about 600, about 660, about 720, about 780, about 840, about 900, about 960, about 1020, about 1080, about 1140, about 1200, about 1260, about 1320, about 1380 or about 1440 minutes and two days. In certain embodiments, the precipitation time is between about 5 minutes and about one day. In certain embodiments, the precipitation time is between about 5 minutes and about 120 minutes.

[0101] The precipitation time can be about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes, about 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes, about 145 minutes, about 150 minutes, about 155 minutes or about 160 minutes.

[0102] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0103] In one embodiment, the optional precipitation step is performed without stirring. In one embodiment, the optional precipitation step is performed with stirring. In another embodiment, the optional precipitation step is performed with gentle stirring. In another embodiment, the optional precipitation step is performed with vigorous stirring.

[0104] In one embodiment of the present invention, the addition of flocculants, precipitation solutions and / or the adjustment of pH are performed at a temperature between about 4°C and about 30°C. In one embodiment, the addition of flocculants, precipitation solutions and / or the adjustment of pH are performed at a temperature of about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C or about 30°C. In one embodiment, the addition of flocculants, precipitation solutions and / or the adjustment of pH are performed at a temperature of about 20°C. The inventors unexpectedly noticed that flocculation can be further improved when performed at high temperatures. Thus, in a particular embodiment of the invention, the addition of a flocculant, a precipitation solution and / or the adjustment of pH is performed at a temperature between about 30° C. and about 95° C. In one embodiment, the addition of a flocculant, a precipitation solution and / or the adjustment of pH is performed at a temperature between about 35° C. and about 80° C., at a temperature between about 40° C. and about 70° C., at a temperature between about 45° C. and about 65° C., at a temperature between about 50° C. and about 60° C., at a temperature between about 50° C. and about 55° C., at a temperature between about 45° C. and about 55° C., or at a temperature between about 45° C. and about 55° C. In one embodiment, the addition of a flocculant, a precipitation solution and / or the adjustment of the pH to about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, The method of the present invention is carried out at a temperature of about 56° C., about 57° C., about 58° C., about 59° C., about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., about 75° C., about 76° C., about 77° C., about 78° C., about 79° C. or about 80° C. In one embodiment, the addition of the flocculant, the precipitation solution and / or the adjustment of the pH are carried out at a temperature of about 50° C.

[0105] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0106] In one embodiment, the addition of the flocculant is performed at any of the above temperatures.

[0107] In one embodiment, the precipitation of the solution after adding the flocculant is performed at any of the above temperatures.

[0108] In one embodiment, the pH adjustment is performed at any of the above temperatures.

[0109] In one embodiment, the adding of the flocculant and the precipitation of the solution after the addition of the flocculant are performed at any of the above temperatures.

[0110] In one embodiment, the addition of flocculant and the adjustment of pH are performed at any of the above temperatures.

[0111] In one embodiment, the addition of the flocculant, the precipitation of the solution after the addition of the flocculant, and the adjustment of the pH are performed at any of the above temperatures.

[0112] In one embodiment, the flocculation step comprises adding a flocculant (as disclosed above) without pH adjustment.

[0113] In one embodiment, the flocculation step comprises adding a flocculant and a precipitation solution (as disclosed above) without pH adjustment.

[0114] In one embodiment, the flocculation step comprises adding a flocculant, adjusting the pH, and precipitating the solution (as disclosed above). In one embodiment, the flocculant is added before adjusting the pH. In another embodiment, the pH is adjusted before adding the flocculant.

[0115] In one embodiment, the flocculation step includes adding a flocculant, a precipitation solution, and adjusting the pH (as disclosed above). In one embodiment, the addition of the flocculant and the precipitation solution is performed before adjusting the pH. In another embodiment, the pH is adjusted before adding the flocculant and the precipitation solution. In one embodiment, the addition of the flocculant and the adjustment of the pH is performed before the precipitation solution. In another embodiment, the pH is adjusted before adding the flocculant and the precipitation solution.

[0116] In one embodiment, the flocculation step includes adding a flocculant, adjusting pH, and adjusting temperature (as disclosed above).

[0117] These steps can be done in any order:

[0118] - Addition of flocculant followed by pH adjustment followed by temperature adjustment, or;

[0119] - Addition of flocculant followed by temperature adjustment followed by pH adjustment, or;

[0120] - pH adjustment followed by addition of flocculant followed by temperature adjustment, or;

[0121] - pH adjustment followed by temperature adjustment followed by addition of flocculant, or;

[0122] - adjusting the temperature, followed by addition of a flocculant, followed by adjustment of the pH, or;

[0123] - Adjust the temperature, then adjust the pH, then add the flocculant.

[0124] Additionally, after addition of the flocculant and / or adjustment of the pH, the solution may be held for a period of time to allow for the precipitation of flocs prior to downstream processing.

[0125] 1.3. Solid / liquid separation

[0126] The flocculated material may be separated from the polysaccharide of interest by any suitable solid / liquid separation method.

[0127] Thus, in one embodiment of the invention, after flocculation, the suspension (as obtained in section 1.2 above) is clarified by decantation, sedimentation, filtration or centrifugation. In one embodiment, the polysaccharide-containing solution is then collected for storage and / or additional processing.

[0128] In one embodiment of the present invention, after flocculation, the suspension (as obtained in section 1.2 above) is clarified by decantation. A decanter is used to separate liquids where the density difference between the liquids is sufficient to cause the floccules to settle. In a decanter in operation, there will be three different zones: clarified heavy liquid, separated dispersed liquid (dispersion zone), and clarified light liquid. In order to produce a clean solution, a small amount of solution must usually be left in the container. The decanter can be designed for continuous operation.

[0129] In one embodiment of the present invention, after flocculation, the suspension (as obtained in section 1.2 above) is clarified by sedimentation (precipitation). Sedimentation is the separation of suspended solid particles from a liquid mixture into a clarified liquid and a slurry with a higher solid content by gravity sedimentation. Sedimentation can be performed in a thickener, clarifier or classifier. Since thickening and clarification are relatively inexpensive processes when used to process large amounts of liquid, they can be used to pre-concentrate the feed for filtration.

[0130] In one embodiment of the present invention, after flocculation, the suspension (as obtained in section 1.2 above) is clarified by centrifugation. In one embodiment, the centrifugation is continuous centrifugation. In one embodiment, the centrifugation is bucket centrifugation. In one embodiment, the supernatant containing the polysaccharide is then collected for storage and / or additional processing.

[0131] In some embodiments, the suspension is about 1,000 g about 2,000 g, about 3,000 g, about 4,000 g, about 5,000 g, about 6,000 g, about 8,000 g, about 9,000 g, about 10,000 g, about 11,000 g, about 12,000 g, about 13,000 g, about 14,000 g, about 15,000 g, about 16,000 g, about 17,000 g, about 18,000 g, about g, about 19,000 g, about 20,000 g, about 25,000 g, about 30,000 g, about 35,000 g, about 40,000 g, about 50,000 g, about 60,000 g, about 70,000 g, about 80,000 g, about 90,000 g, about 100,000 g, about 120,000 g, about 140,000 g, about 160,000 g, or about 180,000 g. In some embodiments, the suspension is centrifuged at about 8,000 g, about 9,000 g, about 10,000 g, about 11,000 g, about 12,000 g, about 13,000 g, about 14,000 g, about 15,000 g, about 16,000 g, about 17,000 g, about 18,000 g, about 19,000 g, about 20,000 g, or about 25,000 g.

[0132] In some embodiments, the suspension is centrifuged at between about 5,000 g and about 25,000 g. In some embodiments, the suspension is centrifuged at between about 8,000 g and about 20,000 g. In some embodiments, the suspension is centrifuged at between about 10,000 g and about 15,000 g. In some embodiments, the suspension is centrifuged at between about 10,000 g and about 12,000 g.

[0133] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0134] In some embodiments, the suspension is centrifuged during at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, or at least 160 minutes. Preferably, the centrifugation time is less than 24 hours.

[0135] Thus in certain embodiments, the suspension is centrifuged for between about 5, about 10, about 15, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 140, about 160, about 180, about 220, about 240, about 300, about 360, about 420, about 480, about 540, about 600, about 660, about 720, about 780, about 840, about 900, about 960, about 1020, about 1080, about 1140, about 1200, about 1260, about 1320, or about 1380 minutes and 1440 minutes.

[0136] Preferably, the suspension is centrifuged between about 5, about 10, about 15, about 20, about 25, about 30, about 60, about 90, about 120, about 180, about 240, about 300, about 360, about 420, about 480, or about 540 minutes and about 600 minutes. In certain embodiments, the suspension is centrifuged between about 5 minutes and about 3 hours. In some cases, the suspension is centrifuged between about 5 minutes and about 120 minutes.

[0137] The suspension may be centrifuged during between about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes, about 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes, about 145 minutes, about 150 minutes, or about 155 minutes and about 160 minutes.

[0138] The suspension may be centrifuged during between about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, or about 55 minutes and about 60 minutes.

[0139] The suspension can be centrifuged during about 5, about 10, about 15, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 140, about 160, about 180, about 220, about 240, about 300, about 360, about 420, about 480, about 540, about 600, about 660, about 720, about 780, about 840, about 900, about 960, about 1020, about 1080, about 1140, about 1200, about 1260, about 1320, about 1380 minutes, or about 1440 minutes.

[0140] The suspension may be centrifuged during about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes, about 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes, about 145 minutes, about 150 minutes, about 155 minutes, or about 160 minutes.

[0141] The suspension may be centrifuged during between about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes.

[0142] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0143] In one embodiment of the invention, centrifugation is continuous centrifugation. In said embodiment, the feed rate may be between 50-5000ml / min, 100-4000ml / min, 150-3000ml / min, 200-2500ml / min, 250-2000ml / min, 300-1500ml / min, 300-1000ml / min, 200-1000ml / min, 200-1500ml / min, 400-1500ml / min, 500-1500ml / min, 500-1000ml / min, 500-2000ml / min, 500-2500ml / min or 1000-2500ml / min.

[0144] In one embodiment, the feed rate may be about 10, about 25, about 50, about 75, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1050, about 1100, about 1150, about 1200, about 1250, about 1300. , about 1350, about 1400, about 1450, about 1500, about 1650, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3250, about 3500, about 3750, about 4000, about 4250, about 4500 or about 5000 ml / min.

[0145] In one embodiment of the present invention, after flocculation, the suspension is clarified by filtration (as obtained in section 1.2 above). In filtration, suspended solid particles in a liquid are removed by passing the mixture through a porous medium, which retains the particles and passes through a clarified filtrate. Filtration is performed on a screen by gravity or on a filter by vacuum, pressure or centrifugation. Solids can be retained on the surface of the filter medium, i.e., cake filtration, or solids can be captured in the filter medium, i.e., deep filtration. In this embodiment, after flocculation, the suspension is clarified by microfiltration (as obtained in section 1.2 above). In one embodiment, the microfiltration is tangential microfiltration. In another embodiment, the microfiltration is dead-end filtration (vertical filtration). In one embodiment, the microfiltration is dead-end filtration, in which diatomaceous earth (DE), also known as DE diatomaceous earth, is used as a filter aid to promote and improve the efficiency of solid / liquid separation. Therefore, in one embodiment, after flocculation, the suspension is clarified by dead-end microfiltration including diatomaceous earth (DE) (as obtained in section 1.2 above). DE can be impregnated (or bonded) into a dead-end filter as part of a depth filter.

[0146] In another form, DE can be added to the flocculation solution in powder form (as obtained after section 1.2). In the latter case, the DE treated flocculation solution can be further clarified by depth filtration.

[0147] In one embodiment, the solution is processed through a microfiltration step, wherein the filter has a nominal retention range between about 0.01-2 microns, about 0.05-2 microns, about 0.1-2 microns, about 0.2-2 microns, about 0.3-2 microns, about 0.4-2 microns, about 0.45-2 microns, about 0.5-2 microns, about 0.6-2 microns, about 0.7-2 microns, about 0.8-2 microns, about 0.9-2 microns, about 1-2 microns, about 1.25-2 microns, about 1.5-2 microns, or about 1.75-2 microns.

[0148] In one embodiment, the solution is processed through a microfiltration step, wherein the filter has a nominal retention range between about 0.01-1 micron, about 0.05-1 micron, about 0.1-1 micron, about 0.2-1 micron, about 0.3-1 micron, about 0.4-1 micron, about 0.45-1 micron, about 0.5-1 micron, about 0.6-1 micron, about 0.7-1 micron, about 0.8-1 micron, or about 0.9-1 micron.

[0149] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0150] In one embodiment, the solution is processed through a microfiltration step wherein the filter has a nominal retention rating of about 0.01, about 0.05, about 0.1, about 0.2, about 0.3, about 0.4, about 0.45, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2 microns.

[0151] In one embodiment, the solution is processed through a microfiltration step wherein the filter has a nominal retention rating of about 0.45 microns.

[0152] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of between 100-5000 L / m 2 、200-5000L / m 2 、300-5000L / m 2 、400-5000L / m 2 , 500-5000L / m 2 、750-5000L / m 2 1000-5000L / m 2 1500-5000L / m 2 , 2000-5000L / m 2 、3000-5000L / m 2 or 4000-5000L / m 2 The filtering capacity between.

[0153] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of between 100-2500 L / m 2 、200-2500L / m 2 、300-2500L / m 2 、400-2500L / m 2 、500-2500L / m 2 、750-2500L / m 2 1000-2500L / m 2 、1500-2500L / m 2 or 2000-2500L / m 2 The filtering capacity between.

[0154] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of between 100-1500 L / m 2 、200-1500L / m 2 、300-1500L / m 2 、400-1500L / m 2 、500-1500L / m 2 、750-1500L / m 2 or 1000-1500L / m 2 The filtering capacity between.

[0155] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of between 100-1250 L / m 2 、200-1250L / m 2 、300-1250L / m 2 、400-1250L / m 2 、500-1250L / m 2 , 750-1250L / m 2 or 1000-1250L / m 2 The filtering capacity between.

[0156] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of between 100-1000 L / m 2 、200-1000L / m 2 、300-1000L / m 2 、400-1000L / m 2 、500-1000L / m 2 or 750-1000L / m 2 The filtering capacity between.

[0157] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of between 100-750 L / m 2 、200-750L / m 2 、300-750L / m 2 、400-750L / m 2 or 500-750L / m 2 The filtering capacity between.

[0158] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of between 100-600 L / m 2 , 200-600L / m 2 、300-600L / m 2 、400-600L / m 2 or 400-600L / m 2 The filtering capacity between.

[0159] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of between 100-500 L / m 2 、200-500L / m 2 、300-500L / m 2 or 400-500L / m 2 The filtering capacity between.

[0160] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0161] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a diameter of about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1050, about 1100, about 1150, about 1200, about 1250, about about 1300, about 1350, about 1400, about 1450, about 1500, about 1550, about 1600, about 1650, about 1700, about 1750, about 1800, about 1850, about 1900, about 1950, about 2000, about 2050, about 2100, about 2150, about 2200, about 2250, about 2300, about 2350, about 2400, about 2450 or about 2500 L / m 2 filtering capacity.

[0162] The above solid / liquid separation methods can be used alone, or in combination of two in any order, or in combination of three in any order.

[0163] 1.4 Filtering (e.g. depth filtering)

[0164] Once the solution has been processed through the flocculation step of section 1.2 above and / or through the solid / liquid separation step of section 1.3 above, the polysaccharide-containing solution (eg, the supernatant) may optionally be further clarified.

[0165] In one embodiment, the solution is filtered to produce a further clarified solution. In one embodiment, filtration is directly applied to a solution obtained by any of the methods in Section 1.2 above. In one embodiment, filtration is applied to a solution further clarified by a solid / liquid separation step as described in Section 1.3 above.

[0166] In one embodiment, the solution is treated by a filtration step selected from the group consisting of: depth filtration, activated carbon filtration, size filtration, diafiltration and ultrafiltration. In one embodiment, the solution is treated by a diafiltration step, in particular by tangential flow filtration. In one embodiment, the solution is treated by a depth filtration step.

[0167] Depth filters use porous filter media to retain particles throughout the media, not just on the surface of the media. Due to the tortuous and channel-like nature of the filter media, particles are retained throughout the media within its structure, not just on the surface.

[0168] In one embodiment, the solution is treated by a depth filtration step, wherein the depth filter design is selected from the group consisting of: a cassette, a cartridge, a deep bed (eg, a sand filter), and a lenticular filter.

[0169] In one embodiment, the solution is processed by a depth filtration step, wherein the depth filter has a diameter between about 0.01-100 microns, about 0.05-100 microns, about 0.1-100 microns, about 0.2-100 microns, about 0.3-100 microns, about 0.4-100 microns, about 0.5-100 microns, about 0.6-100 microns, about 0.7-100 microns, about 0.8-100 microns, about 0.9-100 microns, about 1-100 microns, about 1.25-100 microns, about 1.5 -100 microns, about 1.75-100 microns, about 2-100 microns, about 3-100 microns, about 4-100 microns, about 5-100 microns, about 6-100 microns, about 7-100 microns, about 8-100 microns, about 9-100 microns, about 10-100 microns, about 15-100 microns, about 20-100 microns, about 25-100 microns, about 30-100 microns, about 40-100 microns, about 50-100 microns, or about 75-100 microns.

[0170] In one embodiment, the solution is processed by a depth filtration step, wherein the depth filter has a depth of about 0.01-75 microns, about 0.05-75 microns, about 0.1-75 microns, about 0.2-75 microns, about 0.3-75 microns, about 0.4-75 microns, about 0.5-75 microns, about 0.6-75 microns, about 0.7-75 microns, about 0.8-75 microns, about 0.9-75 microns, about 1-75 microns, about 1.25-75 The nominal retention range is between about 1.5-75 microns, about 1.75-75 microns, about 2-75 microns, about 3-75 microns, about 4-75 microns, about 5-75 microns, about 6-75 microns, about 7-75 microns, about 8-75 microns, about 9-75 microns, about 10-75 microns, about 15-75 microns, about 20-75 microns, about 25-75 microns, about 30-75 microns, about 40-75 microns, or about 50-75 microns.

[0171] In one embodiment, the solution is processed by a depth filtration step, wherein the depth filter has a thickness of about 0.01-50 microns, about 0.05-50 microns, about 0.1-50 microns, about 0.2-50 microns, about 0.3-50 microns, about 0.4-50 microns, about 0.5-50 microns, about 0.6-50 microns, about 0.7-50 microns, about 0.8-50 microns, about 0.9-50 microns, about 1-50 microns, about 1.25-50 The nominal retention range is between about 1.5-50 microns, about 1.75-50 microns, about 2-50 microns, about 3-50 microns, about 4-50 microns, about 5-50 microns, about 6-50 microns, about 7-50 microns, about 8-50 microns, about 9-50 microns, about 10-50 microns, about 15-50 microns, about 20-50 microns, about 25-50 microns, about 30-50 microns, about 40-50 microns, or about 50-50 microns.

[0172] In one embodiment, the solution is processed through a depth filtration step, wherein the depth filter has a nominal retention range between about 0.01-25 microns, about 0.05-25 microns, about 0.1-25 microns, about 0.2-25 microns, about 0.3-25 microns, about 0.4-25 microns, about 0.5-25 microns, about 0.6-25 microns, about 0.7-25 microns, about 0.8-25 microns, about 0.9-25 microns, about 1-25 microns, about 1.25-25 microns, about 1.5-25 microns, about 1.75-25 microns, about 2-25 microns, about 3-25 microns, about 4-25 microns, about 5-25 microns, about 6-25 microns, about 7-25 microns, about 8-25 microns, about 9-25 microns, about 10-25 microns, about 15-25 microns, or about 20-25 microns.

[0173] In one embodiment, the solution is processed through a depth filtration step, wherein the depth filter has a nominal retention range between about 0.01-10 microns, about 0.05-10 microns, about 0.1-10 microns, about 0.2-10 microns, about 0.3-10 microns, about 0.4-10 microns, about 0.5-10 microns, about 0.6-10 microns, about 0.7-10 microns, about 0.8-10 microns, about 0.9-10 microns, about 1-10 microns, about 1.25-10 microns, about 1.5-10 microns, about 1.75-10 microns, about 2-10 microns, about 3-10 microns, about 4-10 microns, about 5-10 microns, about 6-10 microns, about 7-10 microns, about 8-10 microns, or about 9-10 microns.

[0174] In one embodiment, the solution is processed through a depth filtration step, wherein the depth filter has a nominal retention range between about 0.01-8 microns, about 0.05-8 microns, about 0.1-8 microns, about 0.2-8 microns, about 0.3-8 microns, about 0.4-8 microns, about 0.5-8 microns, about 0.6-8 microns, about 0.7-8 microns, about 0.8-8 microns, about 0.9-8 microns, about 1-8 microns, about 1.25-8 microns, about 1.5-8 microns, about 1.75-8 microns, about 2-8 microns, about 3-8 microns, about 4-8 microns, about 5-8 microns, about 6-8 microns, or about 7-8 microns.

[0175] In one embodiment, the solution is processed through a depth filtration step, wherein the depth filter has a nominal retention range between about 0.01-5 microns, about 0.05-5 microns, about 0.1-5 microns, about 0.2-5 microns, about 0.3-5 microns, about 0.4-5 microns, about 0.5-5 microns, about 0.6-5 microns, about 0.7-5 microns, about 0.8-5 microns, about 0.9-5 microns, about 1-5 microns, about 1.25-5 microns, about 1.5-5 microns, about 1.75-5 microns, about 2-5 microns, about 3-5 microns, or about 4-5 microns.

[0176] In one embodiment, the solution is processed through a depth filtration step, wherein the depth filter has a nominal retention range of between about 0.01-2 microns, about 0.05-2 microns, about 0.1-2 microns, about 0.2-2 microns, about 0.3-2 microns, about 0.4-2 microns, about 0.5-2 microns, about 0.6-2 microns, about 0.7-2 microns, about 0.8-2 microns, about 0.9-2 microns, about 1-2 microns, about 1.25-2 microns, about 1.5-2 microns, about 1.75-2 microns, about 2-2 microns, about 3-2 microns, or about 4-2 microns.

[0177] In one embodiment, the solution is processed through a depth filtration step, wherein the depth filter has a nominal retention range between about 0.01-1 micron, about 0.05-1 micron, about 0.1-1 micron, about 0.2-1 micron, about 0.3-1 micron, about 0.4-1 micron, about 0.5-1 micron, about 0.6-1 micron, about 0.7-1 micron, about 0.8-1 micron, or about 0.9-1 micron.

[0178] In one embodiment, the solution is processed through a depth filtration step, wherein the depth filter has a nominal retention range between about 0.05-50 microns, 0.1-25 microns, 0.2-10 microns, 0.1-10 microns, 0.2-5 microns, or 0.25-1 micron.

[0179] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0180] In one embodiment, the solution is treated by a depth filtration step, wherein the depth filter has a flow rate of 1-2500 L / m 2 、5-2500L / m 2 10-2500L / m 2 、25-2500L / m 2 、50-2500L / m 2 、75-2500L / m 2 100-2500L / m 2 150-2500L / m 2 、200-2500L / m 2 、300-2500L / m 2 、400-2500L / m 2 、500-2500L / m 2 、750-2500L / m 2 1000-2500L / m 2 、1500-2500L / m 2 or 2000-2500L / m 2 filtering capacity.

[0181] In one embodiment, the solution is treated by a depth filtration step, wherein the depth filter has a flow rate of 1-1000 L / m 2 、5-1000L / m 2 10-1000L / m 2 、25-1000L / m 2 、50-1000L / m 2 , 75-1000L / m 2 100-1000L / m 2 150-1000L / m2 、200-1000L / m 2 、300-1000L / m 2 、400-1000L / m 2 、500-1000L / m 2 or 750-1000L / m 2 filtering capacity.

[0182] In one embodiment, the solution is treated by a depth filtration step, wherein the depth filter has a flow rate of 1-750 L / m 2 、5-750L / m 2 、10-750L / m 2 、25-750L / m 2 、50-750L / m 2 , 75-750L / m 2 100-750L / m 2 150-750L / m 2 、200-750L / m 2 、300-750L / m 2 、400-750L / m 2 or 500-750L / m 2 filtering capacity.

[0183] In one embodiment, the solution is treated by a depth filtration step, wherein the depth filter has a flow rate of 1-500 L / m 2 、5-500L / m 2 10-500L / m 2 、25-500L / m 2 , 50-500L / m 2 、75-500L / m 2 100-500L / m 2 150-500L / m 2 、200-500L / m 2 、300-500L / m 2 or 400-500L / m 2 filtering capacity.

[0184] In one embodiment, the solution is treated by a depth filtration step, wherein the depth filter has a flow rate of 1-400 L / m 2 、5-400L / m 2 10-400L / m 2 、25-400L / m 2 、50-400L / m 2 、75-400L / m 2 100-400L / m2 、150-400L / m 2 、200-400L / m 2 or 300-400L / m 2 filtering capacity.

[0185] In one embodiment, the solution is treated by a depth filtration step, wherein the depth filter has a flow rate of 1-300 L / m 2 、5-300L / m 2 10-300L / m 2 、25-300L / m 2 、50-300L / m 2 , 75-300L / m 2 100-300L / m 2 、150-300L / m 2 or 200-300L / m 2 filtering capacity.

[0186] In one embodiment, the solution is treated by a depth filtration step, wherein the depth filter has a flow rate of 1-200 L / m 2 、5-200L / m 2 10-200L / m 2 、25-200L / m 2 、50-200L / m 2 , 75-200L / m 2 100-200L / m 2 or 150-200L / m 2 filtering capacity.

[0187] In one embodiment, the solution is treated by a depth filtration step, wherein the depth filter has a flow rate of 1-100 L / m 2 , 5-100L / m 2 10-100L / m 2 、25-100L / m 2 、50-100L / m 2 or 75-100L / m 2 filtering capacity.

[0188] In one embodiment, the solution is treated by a depth filtration step, wherein the depth filter has a flow rate of 1-50 L / m 2 , 5-50L / m 2 10-50L / m 2 or 25-50L / m 2 filtering capacity.

[0189] Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0190] In one embodiment, the solution is treated by a depth filtration step, wherein the feed rate is between 1-1000 LMH (liters / m 2 / hour), 10-1000LMH, 25-1000LMH, 50-1000LMH, 100-1000LMH, 125-1000LMH, 150-1000LMH, 200-1000LMH, 250-1000LMH, 300-1000LMH, 400-1000LMH, 500-1000LMH, 600-1000LMH, 700-1000LMH, 800-1000LMH or 900-1000LMH.

[0191] In one embodiment, the solution is processed through a depth filtration step wherein the feed rate is between 1-500LMH, 10-500LMH, 25-500LMH, 50-500LMH, 100-500LMH, 125-500LMH, 150-500LMH, 200-500LMH, 250-500LMH, 300-500LMH or 400-500LMH.

[0192] In one embodiment, the solution is processed through a depth filtration step wherein the feed rate is between 1-400LMH, 10-400LMH, 25-400LMH, 50-400LMH, 100-400LMH, 125-400LMH, 150-400LMH, 200-400LMH, 250-400LMH, or 300-400LMH.

[0193] In one embodiment, the solution is processed through a depth filtration step wherein the feed rate is between 1-250 LMH, 10-250 LMH, 25-250 LMH, 50-250 LMH, 100-250 LMH, 125-250 LMH, 150-250 LMH, or 200-250 LMH.

[0194] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0195] In one embodiment, the solution is processed through a depth filtration step wherein the feed rate is about 1, about 2, about 5, about 10, about 25, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500 0, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 LMH.

[0196] 1.5 Optional further filtering

[0197] Once the solution has been processed through the filtration step in section 1.4 above, the obtained solution (ie, the filtrate) may optionally be further clarified.

[0198] In one embodiment, the solution is subjected to microfiltration. In one embodiment, the microfiltration is dead-end filtration (vertical filtration). In one embodiment, the microfiltration is tangential microfiltration.

[0199] In one embodiment, the solution is processed through a microfiltration step, wherein the filter has a nominal retention range between about 0.01-2 microns, about 0.05-2 microns, about 0.1-2 microns, about 0.2-2 microns, about 0.3-2 microns, about 0.4-2 microns, about 0.45-2 microns, about 0.5-2 microns, about 0.6-2 microns, about 0.7-2 microns, about 0.8-2 microns, about 0.9-2 microns, about 1-2 microns, about 1.25-2 microns, about 1.5-2 microns, or about 1.75-2 microns.

[0200] In one embodiment, the solution is processed through a depth filtration step, wherein the filter has a nominal retention range between about 0.01-1 micron, about 0.05-1 micron, about 0.1-1 micron, about 0.2-1 micron, about 0.3-1 micron, about 0.4-1 micron, about 0.45-1 micron, about 0.5-1 micron, about 0.6-1 micron, about 0.7-1 micron, about 0.8-1 micron, or about 0.9-1 micron.

[0201] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0202] In one embodiment, the solution is processed through a microfiltration step wherein the filter has a nominal retention rating of about 0.01, about 0.05, about 0.1, about 0.2, about 0.3, about 0.4, about 0.45, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2 microns.

[0203] In one embodiment, the solution is processed through a microfiltration step wherein the filter has a nominal retention rating of about 0.45 microns.

[0204] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of between 100-5000 L / m 2 、200-5000L / m 2 、300-5000L / m 2 、400-5000L / m 2 , 500-5000L / m 2 、750-5000L / m 2 1000-5000L / m 2 1500-5000L / m 2 , 2000-5000L / m 2 、3000-5000L / m 2 or 4000-5000L / m 2 The filtering capacity between.

[0205] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of between 100-2500 L / m 2 、200-2500L / m 2 、300-2500L / m 2 、400-2500L / m 2 、500-2500L / m 2 、750-2500L / m 2 1000-2500L / m 2 、1500-2500L / m 2 or 2000-2500L / m 2 The filtering capacity between.

[0206] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of between 100-1500 L / m 2 、200-1500L / m 2 、300-1500L / m 2 、400-1500L / m 2、500-1500L / m 2 、750-1500L / m 2 or 1000-1500L / m 2 The filtering capacity between.

[0207] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of between 100-1250 L / m 2 、200-1250L / m 2 、300-1250L / m 2 、400-1250L / m 2 、500-1250L / m 2 , 750-1250L / m 2 or 1000-1250L / m 2 The filtering capacity between.

[0208] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of between 100-1000 L / m 2 、200-1000L / m 2 、300-1000L / m 2 、400-1000L / m 2 、500-1000L / m 2 or 750-1000L / m 2 The filtering capacity between.

[0209] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of between 100-750 L / m 2 、200-750L / m 2 、300-750L / m 2 、400-750L / m 2 or 500-750L / m 2 The filtering capacity between.

[0210] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of between 100-600 L / m 2 , 200-600L / m 2 、300-600L / m 2 、400-600L / m 2 or 400-600L / m 2 The filtering capacity between.

[0211] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of between 100-500 L / m 2 、200-500L / m 2、300-500L / m 2 or 400-500L / m 2 The filtering capacity between.

[0212] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0213] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a diameter of about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1050, about 1100, about 1150, about 1200, about 1250, about about 1300, about 1350, about 1400, about 1450, about 1500, about 1550, about 1600, about 1650, about 1700, about 1750, about 1800, about 1850, about 1900, about 1950, about 2000, about 2050, about 2100, about 2150, about 2200, about 2250, about 2300, about 2350, about 2400, about 2450 or about 2500 L / m 2 filtering capacity.

[0214] 1.6 Ultrafiltration and / or Diafiltration

[0215] Once the solution has been filtered by any of the methods in Section 1.4 above and / or by the filtration step in Section 1.5 above, the resulting solution (ie, the filtrate) may optionally be further clarified by ultrafiltration and / or diafiltration.

[0216] Ultrafiltration (UF) is a process for concentrating dilute product streams. UF separates molecules in solution based on membrane pore size or molecular weight cut-off (MWCO).

[0217] In one embodiment of the present invention, the solution (eg, the filtrate obtained in section 1.5 or 1.6 above) is treated by ultrafiltration.

[0218] In one embodiment, the solution is treated by ultrafiltration, and the molecular weight cutoff of the membrane is in the range of about 5kDa-1000kDa. In one embodiment, the molecular weight cutoff of the membrane is in the range of about 10kDa-750kDa. In one embodiment, the molecular weight cutoff of the membrane is in the range of about 10kDa-500kDa. In one embodiment, the molecular weight cutoff of the membrane is in the range of about 10kDa-300kDa. In one embodiment, the molecular weight cutoff of the membrane is in the range of about 10kDa-100kDa. In one embodiment, the molecular weight cutoff of the membrane is in the range of about 10kDa-50kDa. In one embodiment, the molecular weight cutoff of the membrane is in the range of about 10kDa-30kDa. In one embodiment, the molecular weight cutoff of the membrane is in the range of about 5 kDa-1000 kDa, about 10 kDa-1000 kDa, about 20 kDa-1000 kDa, about 30 kDa-1000 kDa, about 40 kDa-1000 kDa, about 50 kDa-1000 kDa, about 75 kDa-1000 kDa, about 100 kDa-1000 kDa, about 150 kDa-1000 kDa, about 200 kDa-1000 kDa, about 300 kDa-1000 kDa, about 400 kDa-1000 kDa, about 500 kDa-1000 kDa, or about 750 kDa-1000 kDa.

[0219] In one embodiment, the molecular weight cutoff of the membrane is in the range of about 5 kDa-500 kDa, about 10 kDa-500 kDa, about 20 kDa-500 kDa, about 30 kDa-500 kDa, about 40 kDa-500 kDa, about 50 kDa-500 kDa, about 75 kDa-500 kDa, about 100 kDa-500 kDa, about 150 kDa-500 kDa, about 200 kDa-500 kDa, about 300 kDa-500 kDa, or about 400 kDa-500 kDa.

[0220] In one embodiment, the molecular weight cutoff of the membrane is in the range of about 5 kDa-300 kDa, about 10 kDa-300 kDa, about 20 kDa-300 kDa, about 30 kDa-300 kDa, about 40 kDa-300 kDa, about 50 kDa-300 kDa, about 75 kDa-300 kDa, about 100 kDa-300 kDa, about 150 kDa-300 kDa, or about 200 kDa-300 kDa.

[0221] In one embodiment, the molecular weight cutoff of the membrane is in the range of about 5 kDa-100 kDa, about 10 kDa-100 kDa, about 20 kDa-100 kDa, about 30 kDa-100 kDa, about 40 kDa-100 kDa, about 50 kDa-100 kDa, or about 75 kDa-100 kDa.

[0222] In one embodiment, the molecular weight cutoff of the membrane is about 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 200 kDa, about 250 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 750 kDa, or about 1000 kDa.

[0223] In one embodiment, the concentration factor of the ultrafiltration step is about 1.5 to 10. In one embodiment, the concentration factor is about 2 to 8. In one embodiment, the concentration factor is about 2 to 5.

[0224] In one embodiment, the concentration factor is about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, or about 10.0. In one embodiment, the concentration factor is about 2, about 3, about 4, about 5, or about 6.

[0225] In one embodiment of the present invention, the solution (eg, the filtrate obtained in section 1.4 or 1.5 above) is treated by diafiltration.

[0226] In one embodiment of the present invention, the solution obtained after ultrafiltration (UF) as disclosed in the above text section is further treated by diafiltration (UF / DF treatment).

[0227] Diafiltration (DF) is used to exchange the product into a desired buffer solution (or just water). In one embodiment, diafiltration is used to change the chemistry of the retention solution at a constant volume. Unwanted particles pass through the membrane while the composition of the feed stream is changed to a more desirable state by adding a replacement solution (buffer solution, saline solution, buffered saline solution, or water).

[0228] In one embodiment, the replacement solution is water.

[0229] In one embodiment, the replacement solution is saline. In some embodiments, the salt is selected from the group consisting of magnesium chloride, potassium chloride, sodium chloride, and combinations thereof. In a specific embodiment, the salt is sodium chloride. In one embodiment, the replacement solution is about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM sodium chloride. In a specific embodiment, the replacement solution is about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 250 mM or about 300 mM sodium chloride.

[0230] In one embodiment, the replacement solution is a buffer solution. In one embodiment, the replacement solution is a buffer solution, wherein the buffer is selected from the group consisting of: N-(2-acetamido)-aminoethanesulfonic acid (ACES), salts of acetic acid (acetate), N-(2-acetamido)-iminodiacetic acid (ADA), 2-aminoethanesulfonic acid (AES, taurine), ammonia, 2-amino-2-methyl-1-propanol (AMP), 2-amino-2-methyl-1,3-propanediol AMPD, aminomethyl propanediol (ammediol), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), N,N-bis-(2-hydroxyethyl)-2-amino 1-Hydroxyethylamino]-1-propanesulfonic acid (BES), sodium bicarbonate (bicarbonate), N,N'-bis(2-hydroxyethyl)-glycine (diglycine), [bis-(2-hydroxyethyl)-imino]-tris-(hydroxymethylmethane) (BIS-Tris), 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BIS-Tris-propane), boric acid, dimethylarsinic acid (dimethylarsinic acid salt), 3-(cyclohexylamino)-propanesulfonic acid (CAPS), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), sodium carbonate (carbonate), cyclohexylaminoethanesulfonic acid (CHES), salts of citric acid (citrate), 3-[N-bis(hydroxyethyl)-imino]-tris-(hydroxymethyl)-1-propanesulfonic acid (Cyclo ...

[0063] N-(2-hydroxyethyl)-piperazine-N'-ethanesulfonic acid (HEPES), N-(2-hydroxyethyl)-piperazine-N'-3-propanesulfonic acid (HEPPS, EPPS), N-(2-hydroxyethyl)-piperazine-N'-2-hydroxypropanesulfonic acid (HEPPSO), imidazole, salts of malic acid (malate), salts of maleic acid (maleate), 2-(N-morpholino)-ethanesulfonic acid (MES), 3-(N-morpholino)-propanesulfonic acid (MOPS), 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO), salts of phosphoric acid (phosphate), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), pyridine, salts of succinic acid (succinate), 3-{[tris(hydroxymethyl)-methyl]-amino}-propanesulfonic acid (TAPS), 3-[N-tris(hydroxymethyl)-methylamino]-2-hydroxypropanesulfonic acid (TAPSO), triethanolamine (TEA), 2-[tris(hydroxymethyl)-methylamino]-ethanesulfonic acid (TES), N-[tris(hydroxymethyl)-methyl]-glycine (tricin), and tris(hydroxymethyl)-aminomethane (Tris).

[0231] In one embodiment, the diafiltration buffer is selected from the group consisting of salts of acetic acid (acetate), salts of citric acid (citrate), salts of formic acid (formates), salts of malic acid (malates), salts of maleic acid (maleates), salts of phosphoric acid (phosphates), and salts of succinic acid (succinates). In one embodiment, the diafiltration buffer is a salt of citric acid (citrate). In one embodiment, the diafiltration buffer is a salt of succinic acid (succinate). In one embodiment, the salt is a sodium salt. In one embodiment, the salt is a potassium salt.

[0232] In one embodiment, the pH of the diafiltration buffer is between about 4.0-11.0, between about 5.0-10.0, between about 5.5-9.0, between about 6.0-8.0, between about 6.0-7.0, between about 6.5-7.5, between about 6.5-7.0, or between about 6.0-7.5. Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0233] In one embodiment, the pH of the diafiltration buffer is about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, or about 11.0. In one embodiment, the pH of the diafiltration buffer is about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. In one embodiment, the pH of the diafiltration buffer is about 6.5, about 7.0, or about 7.5. In one embodiment, the pH of the diafiltration buffer is about 7.0.

[0234] In one embodiment, the concentration of the diafiltration buffer is between about 0.01 mM-100 mM, between about 0.1 mM-100 mM, between about 0.5 mM-100 mM, between about 1 mM-100 mM, between about 2 mM-100 mM, between about 3 mM-100 mM, between about 4 mM-100 mM, between about 5 mM-100 mM, between about 6 mM-100 mM between about 7mM and 100mM, between about 8mM and 100mM, between about 9mM and 100mM, between about 10mM and 100mM, between about 11mM and 100mM, between about 12mM and 100mM, between about 13mM and 100mM, between about 14mM and 100mM, between about 15mM and 100mM, between about 16mM and 100mM, between about 17mM and 100mM, between about 18mM and 100mM, between about 19mM and 100mM, between about 20mM and 100mM, between about 21mM and 100mM, between about 22mM and 100mM, between about 23mM and 100mM, between about 24mM and 100mM, between about 25mM and 100mM, between about 26mM and 100mM, between about 27mM and 100mM, between about 28mM and 100mM, between about 29mM and 100mM, between about 30mM and 100mM, between about 31mM and 100mM, between about 32mM and 100mM, between about 33mM and 100mM, between about 34mM and 100mM, between about 35mM and 100mM, between about 36mM and 100mM, between about 37mM and 100mM, between about 38mM and 100mM, between about 39mM and 100mM, between about 40mM and 100mM, between about 4 mM, between about 17mM-100mM, between about 18mM-100mM, between about 19mM-100mM, between about 20mM-100mM, between about 25mM-100mM, between about 30mM-100mM, between about 35mM-100mM, between about 40mM-100mM, between about 45mM-100mM, between about 50 In some embodiments, the present invention relates to a molecular weight distribution of the present invention and / or a molecular weight distribution of the present invention. The molecular weight distribution of the present invention can be between about 100 mM and about 100 mM, between about 55 mM and about 100 mM, between about 60 mM and about 100 mM, between about 65 mM and about 100 mM, between about 70 mM and about 100 mM, between about 75 mM and about 100 mM, between about 80 mM and about 100 mM, between about 85 mM and about 100 mM, between about 90 mM and about 100 mM, or between about 95 mM and about 100 mM.

[0235] In one embodiment, the concentration of the diafiltration buffer is between about 0.01 mM-50 mM, between about 0.1 mM-50 mM, between about 0.5 mM-50 mM, between about 1 mM-50 mM, between about 2 mM-50 mM, between about 3 mM-50 mM, between about 4 mM-50 mM, between about 5 mM-50 mM, between about 6 mM-50 mM, between about 7 mM-50 mM, between about 8 mM-50 mM, between about 9 mM-50 mM, between about 10 mM-50 mM, between about 11 mM-50 mM, between about 12 mM-50 mM, between about 13 mM-50 mM, between about 14 mM-50 mM, between about 15 mM-50 mM, between about 16 mM-50 mM, between about 17 mM-50 mM, between about 18 mM-50 mM, between about 19 mM-50 mM, between about 20 mM-50 mM, between about 21 mM-50 mM, between about 22 mM-50 mM, between about 24 mM-50 mM, between about 25 mM-50 mM, between about 26 mM-50 mM, between about 27 mM-50 mM, between about 28 mM-50 mM, between about 29 mM-50 mM, between about 30 mM-50 mM, between about 31 mM-50 mM, between about 32 mM-50 mM, between about 34 mM-50 mM, between about 35 mM-50 mM, between about 36 mM-50 mM, between about 37 mM-50 mM, between about 38 mM-5 In some embodiments, the present invention relates to an embodiment of the present invention which is preferably between about 1mM and 50mM, between about 12mM and 50mM, between about 13mM and 50mM, between about 14mM and 50mM, between about 15mM and 50mM, between about 16mM and 50mM, between about 17mM and 50mM, between about 18mM and 50mM, between about 19mM and 50mM, between about 20mM and 50mM, between about 25mM and 50mM, between about 30mM and 50mM, between about 35mM and 50mM, between about 40mM and 50mM, or between about 45mM and 50mM.

[0236] In one embodiment, the concentration of the diafiltration buffer is between about 0.01 mM-25 mM, between about 0.1 mM-25 mM, between about 0.5 mM-25 mM, between about 1 mM-25 mM, between about 2 mM-25 mM, between about 3 mM-25 mM, between about 4 mM-25 mM, between about 5 mM-25 mM, between about 6 mM-25 mM, between about 7 mM-25 mM, between about 8 mM-25 mM, between about 9 mM-25 mM, between about 10 mM-25 mM, between about 11 mM-25 mM, between about 12 mM-25 mM, between about 13 mM-25 mM, between about 14 mM-25 mM, between about 15 mM-25 mM, between about 16 mM-25 mM, between about 17 mM-25 mM, between about 18 mM-25 mM, between about 19 mM-25 mM, between about 20 mM-25 mM, between about 21 mM-25 mM, between about 23 mM-25 mM, between about 24 mM-25 mM, between about 25 mM-25 mM, between about 26 mM-25 mM, between about 27 mM-25 mM, between about 28 mM-25 mM, between about 29 mM-25 mM, between about 30 mM-25 mM, between about 35 mM-25 mM, between about 36 mM-25 mM, between about 37 mM-25 mM, between about 38 mM-25 mM, between about 39 mM-25 mM, between about 40 mM-25 mM, between about 50 mM-2 Between about 9mM-25mM, between about 10mM-25mM, between about 11mM-25mM, between about 12mM-25mM, between about 13mM-25mM, between about 14mM-25mM, between about 15mM-25mM, between about 16mM-25mM, between about 17mM-25mM, between about 18mM-25mM, between about 19mM-25mM or between about 20mM-25mM.

[0237] In one embodiment, the concentration of the diafiltration buffer is between about 0.01 mM-15 mM, between about 0.1 mM-15 mM, between about 0.5 mM-15 mM, between about 1 mM-15 mM, between about 2 mM-15 mM, between about 3 mM-15 mM, between about 4 mM-15 mM, between about 5 mM-15 mM, between about 6 mM-15 mM, between about 7 mM-15 mM, between about 8 mM-15 mM, between about 9 mM-15 mM, between about 10 mM-15 mM, between about 11 mM-15 mM, between about 12 mM-15 mM, between about 13 mM-15 mM, or between about 14 mM-15 mM.

[0238] In one embodiment, the concentration of the diafiltration buffer is between about 0.01mM-10mM, between about 0.1mM-10mM, between about 0.5mM-10mM, between about 1mM-10mM, between about 2mM-10mM, between about 3mM-10mM, between about 4mM-10mM, between about 5mM-10mM, between about 6mM-10mM, between about 7mM-10mM, between about 8mM-10mM, or between about 9mM-10mM.

[0239] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0240] In one embodiment, the concentration of the diafiltration buffer is about 0.01 mM, about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM. or about 100 mM.

[0241] In one embodiment, the concentration of the diafiltration buffer is about 0.1 mM, about 0.2 mM, about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0242] In one embodiment, the concentration of the diafiltration buffer is about 10 mM.

[0243] In one embodiment, the replacement solution includes a chelating agent. In one embodiment, the replacement solution includes an alum chelating agent. In some embodiments, the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (EDTA-OH), hydroxyethylenediaminetriacetic acid (HEDTA), ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid (CyDTA), diethylenetriamine-N,N,N',N",N"-pentaacetic acid (DTPA), 1,3-diaminopropan-2-ol-N,N,N',N'-tetraacetic acid (DPTA-OH), ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid) (EDDHA), ethylenediamine- N,N'-dipropionic acid dihydrochloride (EDDP), ethylenediamine-tetra(methylenesulfonic acid) (EDTPO), nitrilotri(methylenephosphonic acid) (NTPO), iminodiacetic acid (IDA), hydroxyiminodiacetic acid (HIDA), nitrilotriacetic acid (NTP), triethylenetetraamine-hexaacetic acid (TTHA), dimercaptosuccinic acid (DMSA), 2,3-dimercapto-1-propanesulfonic acid (DMPS), alpha lipoic acid (ALA), nitrilotriacetic acid (NTA), thiaminetetrahydrofuranylmethyl disulfide (TTFD), dimercaptopropanol, penicillamine, deferoxamine (DFOA), deferasirox, phosphonates, salts of citric acid (citrate), and combinations of these.

[0244] In some embodiments, the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (EDTA-OH), hydroxyethylenediaminetriacetic acid (HEDTA), ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid (CyDTA), diethylenetriamine-N,N,N',N",N"-pentaacetic acid (DTPA), 1,3-diaminopropan-2-ol-N,N,N',N'-tetraacetic acid (DPTA-OH), ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid) (EDDHA), salts of citric acid (citrate), and combinations of these.

[0245] In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0246] In some embodiments, the chelating agent is a salt of citric acid (citrate). In some embodiments, the chelating agent is sodium citrate.

[0247] Typically, the chelating agent is used at a concentration of 1 to 500 mM. In one embodiment, the concentration of the chelating agent in the surrogate solution is 2 to 400 mM. In one embodiment, the concentration of the chelating agent in the surrogate solution is 10 to 400 mM. In one embodiment, the concentration of the chelating agent in the surrogate solution is 10 to 200 mM. In one embodiment, the concentration of the chelating agent in the surrogate solution is 10 to 100 mM. In one embodiment, the concentration of the chelating agent in the surrogate solution is 10 to 50 mM. In one embodiment, the concentration of the chelating agent in the surrogate solution is 10 to 30 mM.

[0248] In one embodiment, the concentration of the chelating agent in the replacement solution is about 0.01 mM, about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 50 mM, about 51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, mM, about 20mM, about 21mM, about 22mM, about 23mM, about 24mM, about 25mM, about 26mM, about 27mM, about 28mM, about 29mM, about 30mM, about 31mM, about 32mM, about 33mM, about 34mM, about 35mM, about 36mM, about 37mM, about 38mM, about 39mM, about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95 or about 100mM.

[0249] In one embodiment, the concentration of the chelating agent in the replacement solution is about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM or about 100 mM.

[0250] In one embodiment, the concentration of the chelator in the replacement solution is about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM.

[0251] In one embodiment, the diafiltration buffer solution comprises a salt. In some embodiments, the salt is selected from the group consisting of magnesium chloride, potassium chloride, sodium chloride, and combinations thereof. In a specific embodiment, the salt is sodium chloride. In one embodiment, the diafiltration buffer solution comprises about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 mM sodium chloride. In a particular embodiment, the diafiltration buffer solution comprises about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 250, or about 300 mM sodium chloride.

[0252] In one embodiment of the present invention, the number of diavolumes is at least 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50. In one embodiment of the invention, the diafiltration volume number is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95 or about 100. In one embodiment of the present invention, the diafiltration volume number is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14 or about 15.

[0253] In one embodiment of the invention, the ultrafiltration and diafiltration steps are performed at a temperature between about 20° C. and about 90° C. In one embodiment, the ultrafiltration and diafiltration steps are performed at a temperature between about 35° C. and about 80° C., at a temperature between about 40° C. and about 70° C., at a temperature between about 45° C. and about 65° C., at a temperature between about 50° C. and about 60° C., at a temperature between about 50° C. and about 55° C., at a temperature between about 45° C. and about 55° C., or at a temperature between about 45° C. and about 55° C.

[0254] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0255] In one embodiment, the ultrafiltration and diafiltration steps are performed at about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C In one embodiment, the ultrafiltration and diafiltration steps are performed at a temperature of about 50° C., about 51° C., about 52° C., about 53° C., about 54° C., about 55° C., about 56° C., about 57° C., about 58° C., about 59° C., about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., about 75° C., about 76° C., about 77° C., about 78° C., about 79° C., or about 80° C. In one embodiment, the ultrafiltration and diafiltration steps are performed at a temperature of about 50° C.

[0256] In one embodiment of the invention, the diafiltration step is performed at a temperature between about 20° C. and about 90° C. In one embodiment, the diafiltration step is performed at a temperature between about 35° C. and about 80° C., at a temperature between about 40° C. and about 70° C., at a temperature between about 45° C. and about 65° C., at a temperature between about 50° C. and about 60° C., at a temperature between about 50° C. and about 55° C., at a temperature between about 45° C. and about 55° C., or at a temperature between about 45° C. and about 55° C.

[0257] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0258] In one embodiment, the diafiltration step is performed at about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C The diafiltration step is performed at a temperature of about 50° C., about 51° C., about 52° C., about 53° C., about 54° C., about 55° C., about 56° C., about 57° C., about 58° C., about 59° C., about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., about 75° C., about 76° C., about 77° C., about 78° C., about 79° C., or about 80° C. In one embodiment, the diafiltration step is performed at a temperature of about 50° C.

[0259] In one embodiment of the present invention, the ultrafiltration step is performed at a temperature between about 20° C. and about 90° C. In one embodiment, the ultrafiltration step is performed at a temperature between about 35° C. and about 80° C., at a temperature between about 40° C. and about 70° C., at a temperature between about 45° C. and about 65° C., at a temperature between about 50° C. and about 60° C., at a temperature between about 50° C. and about 55° C., at a temperature between about 45° C. and about 55° C., or at a temperature between about 45° C. and about 55° C. Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0260] In one embodiment, the ultrafiltration step is performed at about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C. , about 50° C., about 51° C., about 52° C., about 53° C., about 54° C., about 55° C., about 56° C., about 57° C., about 58° C., about 59° C., about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., about 75° C., about 76° C., about 77° C., about 78° C., about 79° C., or about 80° C. In one embodiment, the ultrafiltration step is performed at a temperature of about 50° C.

[0261] 1.7 Activated carbon filtration

[0262] Once the solution has been processed through the flocculation step in section 1.2 above, the polysaccharide-containing solution may optionally be further clarified by an activated carbon filtration step.

[0263] In one embodiment, the solution (e.g., supernatant) of section 1.2 further processed by the solid / liquid separation step of section 1.3 is further clarified by an activated carbon filtration step. In one embodiment, the solution further filtered by any method of section 1.4 above and / or by the filtration step of section 1.5 above is further clarified by an activated carbon filtration step. In one embodiment, the solution further clarified by the ultrafiltration and / or diafiltration step of section 1.6 above is further clarified by an activated carbon filtration step.

[0264] An activated carbon filtration step allows for further removal of host cell impurities such as proteins and nucleic acids as well as colored impurities (see WO 2008 / 118752).

[0265] In one embodiment, activated carbon (also called activated charcoal) is added to the solution in an amount sufficient to absorb most of the protein and nucleic acid contaminants, and then the contaminants are removed after being adsorbed onto the activated carbon. In one embodiment, the activated carbon is added in the form of a powder, a granular carbon bed, a pressed carbon block, or an extruded carbon block (see, for example, Norit activated carbon). In one embodiment, the activated carbon is added in an amount of about 0.1% to 20% (weight volume), 1% to 15% (weight volume), 1% to 10% (weight volume), 2% to 10% (weight volume), 3% to 10% (weight volume), 4% to 10% (weight volume), 5% to 10% (weight volume), 1% to 5% (weight volume) or 2% to 5% (weight volume). The mixture is then stirred and allowed to stand. In one embodiment, the mixture is allowed to stand for about 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, 240 minutes or longer. The activated carbon is then removed. The activated carbon may be removed, for example, by centrifugation or filtration.

[0266] In a preferred embodiment, the solution is filtered through activated carbon fixed in a matrix. The matrix can be any porous filter medium that the solution is permeable. The matrix can include a carrier material and / or a binder material. The carrier material can be a synthetic polymer or a polymer of natural origin. Suitable synthetic polymers can include polystyrene, polyacrylamide and polymethyl methacrylate, while polymers of natural origin can include cellulose, polysaccharides and dextran, agarose. Typically, the polymer carrier material is in the form of a fiber network to provide mechanical rigidity. The binder material can be a resin. The matrix can have the form of a diaphragm. In one embodiment, the activated carbon fixed in the matrix is ​​in the form of a flow-through carbon box. The box is an independent entity containing powdered activated carbon fixed in the matrix and prepared in the form of a diaphragm. The diaphragm can be captured in a plastic permeable support to form a disc.

[0267] Alternatively, the membrane may be spirally wound. To increase the filter surface area, several discs may be stacked on top of each other. In particular, the stacked discs have a central core tube for collecting the carbon treated sample and removing it from the filter. The stacked discs may be in the shape of a lens.

[0268] The activated carbon in carbon filters can come from different raw materials, such as peat, lignite, wood or coconut shells.

[0269] Any process known in the art, such as steam or chemical treatment, can be used to activate the carbon (eg, wood-based phosphoric acid activated carbon).

[0270] In the present invention, the activated carbon fixed in the matrix can be placed in a housing to form an independent filter unit. Each filter unit has its own inlet and outlet for the solution to be purified. Examples of filter units that can be used for the present invention are carbon boxes from Cuno Inc. (Meriden, USA) or Pall Corporation (East Hill, USA). In particular, CUNO zetacarbon filters are suitable for the present invention. These carbon filters include a cellulose matrix, in which activated carbon powder is wrapped and resin bonded in place.

[0271] In one embodiment, the activated carbon filter disclosed above has a diameter between about 0.01-100 microns, about 0.05-100 microns, about 0.1-100 microns, about 0.2-100 microns, about 0.3-100 microns, about 0.4-100 microns, about 0.5-100 microns, about 0.6-100 microns, about 0.7-100 microns, about 0.8-100 microns, about 0.9-100 microns, about 1-100 microns, about 1.25-100 microns, about 1. ... The present invention relates to a nominal micron rating between about 100 microns, about 200 microns, about 150 microns, about 200 microns, about 300 microns, about 400 microns, about 500 microns, about 600 microns, about 700 microns, about 800 microns, about 900 microns, about 1000 microns, about 1500 microns, about 2000 microns, about 2500 microns, about 3000 microns, about 4000 microns, about 5000 microns, or about 7500 microns.

[0272] In one embodiment, the activated carbon filter disclosed above has a range of about 0.01-50 microns, about 0.05-50 microns, about 0.1-50 microns, about 0.2-50 microns, about 0.3-50 microns, about 0.4-50 microns, about 0.5-50 microns, about 0.6-50 microns, about 0.7-50 microns, about 0.8-50 microns, about 0.9-50 microns, about 1-50 microns, about 1.25-50 microns, about Nominal micron ratings between 1.5-50 microns, about 1.75-50 microns, about 2-50 microns, about 3-50 microns, about 4-50 microns, about 5-50 microns, about 6-50 microns, about 7-50 microns, about 8-50 microns, about 9-50 microns, about 10-50 microns, about 15-50 microns, about 20-50 microns, about 25-50 microns, about 30-50 microns, about 40-50 microns, or about 50-50 microns.

[0273] In one embodiment, the activated carbon filter disclosed above has a nominal micron rating between about 0.01-25 microns, about 0.05-25 microns, about 0.1-25 microns, about 0.2-25 microns, about 0.3-25 microns, about 0.4-25 microns, about 0.5-25 microns, about 0.6-25 microns, about 0.7-25 microns, about 0.8-25 microns, about 0.9-25 microns, about 1-25 microns, about 1.25-25 microns, about 1.5-25 microns, about 1.75-25 microns, about 2-25 microns, about 3-25 microns, about 4-25 microns, about 5-25 microns, about 6-25 microns, about 7-25 microns, about 8-25 microns, about 9-25 microns, about 10-25 microns, about 15-25 microns, or about 20-25 microns.

[0274] In one embodiment, the activated carbon filter disclosed above has a nominal micron rating between about 0.01-10 microns, about 0.05-10 microns, about 0.1-10 microns, about 0.2-10 microns, about 0.3-10 microns, about 0.4-10 microns, about 0.5-10 microns, about 0.6-10 microns, about 0.7-10 microns, about 0.8-10 microns, about 0.9-10 microns, about 1-10 microns, about 1.25-10 microns, about 1.5-10 microns, about 1.75-10 microns, about 2-10 microns, about 3-10 microns, about 4-10 microns, about 5-10 microns, about 6-10 microns, about 7-10 microns, about 8-10 microns, or about 9-10 microns.

[0275] In one embodiment, the activated carbon filter disclosed above has a nominal micron rating between about 0.01-8 microns, about 0.05-8 microns, about 0.1-8 microns, about 0.2-8 microns, about 0.3-8 microns, about 0.4-8 microns, about 0.5-8 microns, about 0.6-8 microns, about 0.7-8 microns, about 0.8-8 microns, about 0.9-8 microns, about 1-8 microns, about 1.25-8 microns, about 1.5-8 microns, about 1.75-8 microns, about 2-8 microns, about 3-8 microns, about 4-8 microns, about 5-8 microns, about 6-8 microns or about 7-8 microns.

[0276] In one embodiment, the activated carbon filter disclosed above has a nominal micron rating between about 0.01-5 microns, about 0.05-5 microns, about 0.1-5 microns, about 0.2-5 microns, about 0.3-5 microns, about 0.4-5 microns, about 0.5-5 microns, about 0.6-5 microns, about 0.7-5 microns, about 0.8-5 microns, about 0.9-5 microns, about 1-5 microns, about 1.25-5 microns, about 1.5-5 microns, about 1.75-5 microns, about 2-5 microns, about 3-5 microns or about 4-5 microns.

[0277] In one embodiment, the activated carbon filter disclosed above has a nominal micron rating between about 0.01-2 microns, about 0.05-2 microns, about 0.1-2 microns, about 0.2-2 microns, about 0.3-2 microns, about 0.4-2 microns, about 0.5-2 microns, about 0.6-2 microns, about 0.7-2 microns, about 0.8-2 microns, about 0.9-2 microns, about 1-2 microns, about 1.25-2 microns, about 1.5-2 microns, about 1.75-2 microns, about 2-2 microns, about 3-2 microns or about 4-2 microns.

[0278] In one embodiment, the activated carbon filter disclosed above has a nominal micron rating between about 0.01-1 micron, about 0.05-1 micron, about 0.1-1 micron, about 0.2-1 micron, about 0.3-1 micron, about 0.4-1 micron, about 0.5-1 micron, about 0.6-1 micron, about 0.7-1 micron, about 0.8-1 micron or about 0.9-1 micron.

[0279] In an embodiment, the activated carbon filter disclosed above has a nominal micron rating between about 0.05-50 microns, 0.1-25 microns, 0.2-10 microns, 0.1-10 microns, 0.2-5 microns, or 0.25-1 micron.

[0280] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0281] In one embodiment, the activated carbon filtration step is performed at a feed rate between 1-500LMH, 10-500LMH, 15-500LMH, 20-500LMH, 25-500LMH, 30-500LMH, 40-500LMH, 50-500LMH, 100-500LMH, 125-500LMH, 150-500LMH, 200-500LMH, 250-500LMH, 300-500LMH or 400-500LMH.

[0282] In one embodiment, the activated carbon filtration step is performed at a feed rate between 1-200LMH, 10-200LMH, 15-200LMH, 20-200LMH, 25-200LMH, 30-200LMH, 40-200LMH, 50-200LMH, 100-200LMH, 125-200LMH or 150-200LMH.

[0283] In one embodiment, the activated carbon filtration step is performed at a feed rate between 1-150LMH, 10-150LMH, 15-150LMH, 20-150LMH, 25-150LMH, 30-150LMH, 40-150LMH, 50-150LMH, 100-150LMH or 125-150LMH.

[0284] In one embodiment, the activated carbon filtration step is performed at a feed rate between 1-100 LMH, 10-100 LMH, 15-100 LMH, 20-100 LMH, 25-100 LMH, 30-100 LMH, 40-100 LMH, or 50-100 LMH.

[0285] In one embodiment, the activated carbon filtration step is performed at a feed rate between 1-75LMH, 5-75LMH, 10-75LMH, 15-75LMH, 20-75LMH, 25-75LMH, 30-75LMH, 35-75LMH, 40-75LMH, 45-75LMH, 50-75LMH, 55-75LMH, 60-75LMH, 65-75LMH or 70-75LMH.

[0286] In one embodiment, the activated carbon filtration step is performed at a feed rate between 1-50LMH, 5-50LMH, 7-50LMH, 10-50LMH, 15-50LMH, 20-50LMH, 25-50LMH, 30-50LMH, 35-50LMH, 40-50LMH or 45-50LMH.

[0287] Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0288] In one embodiment, the activated carbon filtration step is performed at a feed rate of about 1, about 2, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 225, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 700, about 800, about 900, about 950 or about 1000 LMH.

[0289] In one embodiment, the solution is processed through an activated carbon filter, wherein the filter has a flow rate of between 5-1000 L / m 2 、10-750L / m 2 15-500L / m 2 、20-400L / m 2 、25-300L / m 2 、30-250L / m 2 、40-200L / m 2 or 30-100L / m 2 The filtering capacity between.

[0290] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0291] In one embodiment, the solution is processed through an activated carbon filter, wherein the filter has a flow rate of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 L / m 2 filtering capacity.

[0292] If the pollutant content is above a fixed threshold after the first activated carbon filtration step, the steps may be repeated. In one embodiment of the invention, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 activated carbon filtration steps are performed. In one embodiment of the invention, 1, 2 or 3 activated carbon filtration steps are performed. In one embodiment of the invention, 1 or 2 activated carbon filtration steps are performed.

[0293] In one embodiment, the solution is processed through a continuous activated carbon filter. In one embodiment, the solution is processed through 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 continuous activated carbon filters. In one embodiment, the solution is processed through 2, 3, 4 or 5 continuous activated carbon filters. In one embodiment, the solution is processed through 2 continuous activated carbon filters. In one embodiment, the solution is processed through 3 continuous activated carbon filters. In one embodiment, the solution is processed through 4 continuous activated carbon filters. In one embodiment, the solution is processed through 5 continuous activated carbon filters.

[0294] In one embodiment, the activated carbon filtration step is performed in a single pass mode.

[0295] In another embodiment, the activated carbon filtration step is performed in a recirculation mode. In such embodiments (recirculation mode), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 activated carbon filtration cycles are performed. In another embodiment, 2, 3, 4, 5, 6, 7, 8, 9 or 10 activated carbon filtration cycles are performed. In one embodiment, 2 or 3 activated carbon filtration cycles are performed. In one embodiment, 2 activated carbon filtration cycles are performed.

[0296] 1.8 Optional further filtering

[0297] Once the solution has been treated through the activated carbon step in Section 1.7 above, the resulting solution (ie, the filtrate) may optionally be further filtered.

[0298] In one embodiment, the solution is subjected to microfiltration. In one embodiment, the microfiltration is dead-end filtration (vertical filtration).

[0299] In one embodiment, the solution is processed through a microfiltration step, wherein the filter has a nominal retention range between about 0.01-2 microns, about 0.05-2 microns, about 0.1-2 microns, about 0.2-2 microns, about 0.3-2 microns, about 0.4-2 microns, about 0.45-2 microns, about 0.5-2 microns, about 0.6-2 microns, about 0.7-2 microns, about 0.8-2 microns, about 0.9-2 microns, about 1-2 microns, about 1.25-2 microns, about 1.5-2 microns, or about 1.75-2 microns.

[0300] In one embodiment, the solution is processed through a microfiltration step, wherein the filter has a nominal retention range between about 0.01-1 micron, about 0.05-1 micron, about 0.1-1 micron, about 0.2-1 micron, about 0.3-1 micron, about 0.4-1 micron, about 0.45-1 micron, about 0.5-1 micron, about 0.6-1 micron, about 0.7-1 micron, about 0.8-1 micron, or about 0.9-1 micron.

[0301] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0302] In one embodiment, the solution is processed through a microfiltration step wherein the filter has a nominal retention rating of about 0.01, about 0.05, about 0.1, about 0.2, about 0.3, about 0.4, about 0.45, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.0 microns.

[0303] In one embodiment, the solution is processed through a microfiltration step wherein the filter has a nominal retention rating of about 0.2 microns.

[0304] In one embodiment, the solution is processed through a microfiltration step, wherein the filter has a filtration capacity of 100-6000 L / m 2 、200-6000L / m 2 、300-6000L / m 2 、400-6000L / m 2 、500-6000L / m 2 、750-6000L / m 21000-6000L / m 2 1500-6000L / m 2 、2000-6000L / m 2 、3000-6000L / m 2 or 4000-6000L / m 2 filtering capacity.

[0305] In one embodiment, the solution is processed through a microfiltration step, wherein the filter has a filtration capacity of 100-4000 L / m 2 、200-4000L / m 2 、300-4000L / m 2 , 400-4000L / m 2 、500-4000L / m 2 , 750-4000L / m 2 1000-4000L / m 2 、1500-4000L / m 2 、2000-4000L / m 2 、2500-4000L / m 2 、3000-4000L / m 2 、3000-4000L / m 2 or 3500-4000L / m 2 filtering capacity.

[0306] In one embodiment, the solution is processed through a microfiltration step, wherein the filter has a flow rate of 100-3750 L / m 2 、200-3750L / m 2 、300-3750L / m 2 、400-3750L / m 2 、500-3750L / m 2 、750-3750L / m 2 、1000-3750L / m 2 、1500-3750L / m 2 、2000-3750L / m 2 、2500-3750L / m 2 、3000-3750L / m 2 、3000-3750L / m 2 or 3500-3750L / m 2 filtering capacity.

[0307] In one embodiment, the solution is processed through a microfiltration step, wherein the filter has a flow rate of 100-1250 L / m 2、200-1250L / m 2 、300-1250L / m 2 、400-1250L / m 2 、500-1250L / m 2 , 750-1250L / m 2 or 1000-1250L / m 2 filtering capacity.

[0308] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0309] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a diameter of about 100, about 200, about 300, about 400, about 550, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600 0, about 2700, about 2800, about 2900, about 3000, about 3100, about 3200, about 3300, about 3400, about 3500, about 3600, about 3700, about 3800, about 3900, about 4000, about 4100, about 4200, about 4300, about 4400, about 4500, about 4600, about 4700, about 4800, about 4900, about 5000, about 5250, about 5500, about 5750 or about 6000 L / m 2 filtering capacity.

[0310] 1.9 Hydrophobic Interaction Chromatography (HIC)

[0311] This unit operation removes any impurities with hydrophobic properties, such as lipopolysaccharides (endotoxins) remaining from previous purification steps.

[0312] Once the solution has been processed through the flocculation step in section 1.2 above, the polysaccharide-containing solution may optionally be further purified through a HIC step.

[0313] In one embodiment, the solution (e.g., supernatant) of section 1.2 is further purified by the HIC step after further processing by the solid / liquid separation step of section 1.3. In one embodiment, the solution further filtered by any of the methods of section 1.5 above and / or by the filtration step of section 1.4 above is further purified by the HIC step. In one embodiment, the solution further clarified by the ultrafiltration and / or diafiltration step of section 1.6 above is further purified by the HIC step. In one embodiment, the solution further clarified by the activated carbon filtration step of section 1.7 is further purified by the HIC step.

[0314] In one embodiment, the solution further clarified by the ultrafiltration and / or diafiltration step in Section 1.6 above is further purified by an ion exchange membrane (IEX) filtration step, and may then be further purified by a HIC step.

[0315] In one embodiment, the HIC step is performed using a hydrophobic adsorbent selected from but not limited to the group consisting of phenyl membranes, butyl-, phenyl- and octyl-agarose, butyl-, phenyl-, ether-, polypropylene glycol- and hexyl organic polymer resins.

[0316] In one embodiment, the hydrophobic adsorbent used in the HIC step is a phenyl membrane, such as SARTOBIND phenyl membrane or CYTIVA's phenyl adsorbent membrane.

[0317] In one embodiment, the hydrophobic adsorbent used in the HIC step is a SARTOBIND phenyl membrane.

[0318] In one embodiment, the material from the previous step (e.g., carbon filtrate) is treated with an equilibration buffer to obtain an operating buffer comprising the material to be purified and a desired salt concentration. In one embodiment, the equilibration buffer comprises salt, and the final salt concentration (i.e., in the operating buffer) is selected from about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2 , about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0 M. In one embodiment, the operating buffer has a pH between about 4.0 and about 8.0. In one embodiment, the pH of the operating buffer is about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0. In one embodiment, the equilibration buffer comprises a salt selected from the group consisting of ammonium sulfate (preferably at a final concentration of 0.5M-3.0M in the operating buffer and pH 6.0±2.0), sodium phosphate (preferably at a final concentration of 0.5M-3.0M in the operating buffer and pH 7.0±1.5), potassium phosphate (preferably at a final concentration of 0.5M-3.0M in the operating buffer and pH 7.0±1.5), sodium sulfate (preferably at a final concentration of 0.1M-0.75M in the operating buffer and pH 6.0±2.0), sodium citrate (preferably at a final concentration of 0.1M-1.5M in the operating buffer and pH 6.0±2.0), or sodium chloride (preferably at a final concentration of 0.5M-5.0M in the operating buffer and pH 7.0±1.5).

[0319] In one embodiment, the equilibration buffer comprises ammonium sulfate, and the final salt concentration in the operating buffer is between about 1.0 M and about 2.0 M, preferably about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0 M).

[0320] In one embodiment, the hydrophobic adsorbent is equilibrated with a running buffer and the material to be purified is then run through the column or membrane in the running buffer.

[0321] In one embodiment, the hydrophobic adsorbent is a phenyl membrane and the flow rate is between about 0.1 and about 20 membrane volumes per minute, about 0.1 and about 10 membrane volumes per minute, about 0.2 and about 10 membrane volumes per minute, about 0.2 and about 5 membrane volumes per minute, about 0.1 and about 1 membrane volume per minute. In one embodiment, the hydrophobic adsorbent is a phenyl membrane and the flow rate is between about 0.1 and about 1.0 membrane volume per minute, preferably about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 membrane volume per minute.

[0322] In one embodiment, the HIC membrane is then rinsed with operating buffer and may be further washed with water. The flow-through is collected together with the buffer rinse as the HIC filtrate, and the water wash is also collected for analysis.

[0323] 1.10 Ultrafiltration / diafiltration

[0324] Once the solution has been processed through the HIC step in Section 1.9 above and / or through the further filtration step in Section 1.8 above, the resulting solution (ie, the filtrate) may optionally be further clarified by ultrafiltration and / or diafiltration.

[0325] In one embodiment of the present invention, the solution (eg obtained in section 1.9 or 1.8 above) is treated by ultrafiltration.

[0326] In one embodiment, the solution is treated by ultrafiltration, and the molecular weight cutoff of the membrane is in the range of about 5kDa-1000kDa. In one embodiment, the molecular weight cutoff of the membrane is in the range of about 10kDa-750kDa. In one embodiment, the molecular weight cutoff of the membrane is in the range of about 10kDa-500kDa. In one embodiment, the molecular weight cutoff of the membrane is in the range of about 10kDa-300kDa. In one embodiment, the molecular weight cutoff of the membrane is in the range of about 10kDa-100kDa. In one embodiment, the molecular weight cutoff of the membrane is in the range of about 10kDa-50kDa. In one embodiment, the molecular weight cutoff of the membrane is in the range of about 10kDa-30kDa. In one embodiment, the molecular weight cutoff of the membrane is in the range of about 5 kDa-1000 kDa, about 10 kDa-1000 kDa, about 20 kDa-1000 kDa, about 30 kDa-1000 kDa, about 40 kDa-1000 kDa, about 50 kDa-1000 kDa, about 75 kDa-1000 kDa, about 100 kDa-1000 kDa, about 150 kDa-1000 kDa, about 200 kDa-1000 kDa, about 300 kDa-1000 kDa, about 400 kDa-1000 kDa, about 500 kDa-1000 kDa, or about 750 kDa-1000 kDa.

[0327] In one embodiment, the molecular weight cutoff of the membrane is in the range of about 5 kDa-500 kDa, about 10 kDa-500 kDa, about 20 kDa-500 kDa, about 30 kDa-500 kDa, about 40 kDa-500 kDa, about 50 kDa-500 kDa, about 75 kDa-500 kDa, about 100 kDa-500 kDa, about 150 kDa-500 kDa, about 200 kDa-500 kDa, about 300 kDa-500 kDa, or about 400 kDa-500 kDa.

[0328] In one embodiment, the molecular weight cutoff of the membrane is in the range of about 5 kDa-300 kDa, about 10 kDa-300 kDa, about 20 kDa-300 kDa, about 30 kDa-300 kDa, about 40 kDa-300 kDa, about 50 kDa-300 kDa, about 75 kDa-300 kDa, about 100 kDa-300 kDa, about 150 kDa-300 kDa, or about 200 kDa-300 kDa.

[0329] In one embodiment, the molecular weight cutoff of the membrane is in the range of about 5 kDa-100 kDa, about 10 kDa-100 kDa, about 20 kDa-100 kDa, about 30 kDa-100 kDa, about 40 kDa-100 kDa, about 50 kDa-100 kDa, or about 75 kDa-100 kDa.

[0330] In one embodiment, the molecular weight cutoff of the membrane is about 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 200 kDa, about 250 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 750 kDa, or about 1000 kDa.

[0331] In one embodiment, the concentration factor of the ultrafiltration step is about 1.5 to about 10.0. In one embodiment, the concentration factor is about 2.0 to about 8.0. In one embodiment, the concentration factor is about 2.0 to about 5.0.

[0332] In one embodiment, the concentration factor is about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, or about 10.0. In one embodiment, the concentration factor is about 2.0, about 3.0, about 4.0, about 5.0, or about 6.0.

[0333] In one embodiment of the present invention, the solution (eg, the filtrate obtained in section 1.9 or 1.8 above) is treated by diafiltration.

[0334] In one embodiment of the present invention, the solution obtained after ultrafiltration (UF) as disclosed in the above text section is further treated by diafiltration (UF / DF treatment).

[0335] Diafiltration (DF) is used to exchange the product into a desired buffer solution (or just water). In one embodiment, diafiltration is used to change the chemistry of the retention solution at a constant volume. Unwanted particles pass through the membrane while the composition of the feed stream is changed to a more desirable state by adding a replacement solution (buffer solution, saline solution, buffered saline solution, or water).

[0336] In one embodiment, the replacement solution is water.

[0337] In one embodiment, the replacement solution is saline. In some embodiments, the salt is selected from the group consisting of magnesium chloride, potassium chloride, sodium chloride, and combinations thereof. In a specific embodiment, the salt is sodium chloride. In one embodiment, the replacement solution is about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 mM sodium chloride. In a specific embodiment, the replacement solution is about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 250, or about 300 mM sodium chloride. In a specific embodiment, the replacement solution is about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 80, about 90, or about 100 mM sodium chloride.

[0338] In one embodiment, the replacement solution is a buffer solution. In one embodiment, the replacement solution is a buffer solution, wherein the buffer is selected from the group consisting of: N-(2-acetamido)-aminoethanesulfonic acid (ACES), salts of acetic acid (acetate), N-(2-acetamido)-iminodiacetic acid (ADA), 2-aminoethanesulfonic acid (AES, taurine), ammonia, 2-amino-2-methyl-1-propanol (AMP), 2-amino-2-methyl-1,3-propanediol AMPD, aminomethyl propanediol (ammediol), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), N,N-bis-(2-hydroxyethyl)-2-amino 1-Hydroxyethylamino]-1-propanesulfonic acid (BES), sodium bicarbonate (bicarbonate), N,N'-bis(2-hydroxyethyl)-glycine (diglycine), [bis-(2-hydroxyethyl)-imino]-tris-(hydroxymethylmethane) (BIS-Tris), 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BIS-Tris-propane), boric acid, dimethylarsinic acid (dimethylarsinic acid salt), 3-(cyclohexylamino)-propanesulfonic acid (CAPS), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), sodium carbonate (carbonate), cyclohexylaminoethanesulfonic acid (CHES), salts of citric acid (citrate), 3-[N-bis(hydroxyethyl)-imino]-tris-(hydroxymethyl)-1-propanesulfonic acid (Cyclo ...

[0063] N-(2-hydroxyethyl)-piperazine-N'-ethanesulfonic acid (HEPES), N-(2-hydroxyethyl)-piperazine-N'-3-propanesulfonic acid (HEPPS, EPPS), N-(2-hydroxyethyl)-piperazine-N'-2-hydroxypropanesulfonic acid (HEPPSO), imidazole, salts of malic acid (malate), salts of maleic acid (maleate), 2-(N-morpholino)-ethanesulfonic acid (MES), 3-(N-morpholino)-propanesulfonic acid (MOPS), 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO), salts of phosphoric acid (phosphate), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), pyridine, salts of succinic acid (succinate), 3-{[tris(hydroxymethyl)-methyl]-amino}-propanesulfonic acid (TAPS), 3-[N-tris(hydroxymethyl)-methylamino]-2-hydroxypropanesulfonic acid (TAPSO), triethanolamine (TEA), 2-[tris(hydroxymethyl)-methylamino]-ethanesulfonic acid (TES), N-[tris(hydroxymethyl)-methyl]-glycine (tricin), and tris(hydroxymethyl)-aminomethane (Tris).

[0339] In one embodiment, the diafiltration buffer is selected from the group consisting of salts of acetic acid (acetate), salts of citric acid (citrate), salts of formic acid (formates), salts of malic acid (malates), salts of maleic acid (maleates), salts of phosphoric acid (phosphates), and salts of succinic acid (succinates). In one embodiment, the diafiltration buffer is a salt of citric acid (citrate). In one embodiment, the diafiltration buffer is a salt of succinic acid (succinate). In one embodiment, the diafiltration buffer is a salt of phosphoric acid (phosphate). In one embodiment, the salt is a sodium salt. In one embodiment, the salt is a potassium salt.

[0340] In one embodiment, the pH of the diafiltration buffer is between about 4.0-11.0, between about 5.0-10.0, between about 5.5-9.0, between about 6.0-8.0, between about 6.0-7.0, between about 6.5-7.5, between about 6.5-7.0, or between about 6.0-7.5. Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0341] In one embodiment, the pH of the diafiltration buffer is about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, or about 11.0. In one embodiment, the pH of the diafiltration buffer is about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. In one embodiment, the pH of the diafiltration buffer is about about 6.5, about 7.0, or about 7.5. In one embodiment, the pH of the diafiltration buffer is about 6.0. In one embodiment, the pH of the diafiltration buffer is about 6.5. In one embodiment, the pH of the diafiltration buffer is about 7.0.

[0342] In one embodiment, the concentration of the diafiltration buffer is between about 0.01 mM-100 mM, between about 0.1 mM-100 mM, between about 0.5 mM-100 mM, between about 1 mM-100 mM, between about 2 mM-100 mM, between about 3 mM-100 mM, between about 4 mM-100 mM, between about 5 mM-100 mM, between about 6 mM-100 mM between about 7mM and 100mM, between about 8mM and 100mM, between about 9mM and 100mM, between about 10mM and 100mM, between about 11mM and 100mM, between about 12mM and 100mM, between about 13mM and 100mM, between about 14mM and 100mM, between about 15mM and 100mM, between about 16mM and 100mM, between about 17mM and 100mM, between about 18mM and 100mM, between about 19mM and 100mM, between about 20mM and 100mM, between about 21mM and 100mM, between about 22mM and 100mM, between about 23mM and 100mM, between about 24mM and 100mM, between about 25mM and 100mM, between about 26mM and 100mM, between about 27mM and 100mM, between about 28mM and 100mM, between about 29mM and 100mM, between about 30mM and 100mM, between about 31mM and 100mM, between about 32mM and 100mM, between about 33mM and 100mM, between about 34mM and 100mM, between about 35mM and 100mM, between about 36mM and 100mM, between about 37mM and 100mM, between about 38mM and 100mM, between about 39mM and 100mM, between about 40mM and 100mM, between about 4 mM, between about 17mM-100mM, between about 18mM-100mM, between about 19mM-100mM, between about 20mM-100mM, between about 25mM-100mM, between about 30mM-100mM, between about 35mM-100mM, between about 40mM-100mM, between about 45mM-100mM, between about 50 In some embodiments, the present invention relates to a molecular weight distribution of the present invention and / or a molecular weight distribution of the present invention. The molecular weight distribution of the present invention can be between about 100 mM and about 200 mM, about 55 mM and about 100 mM, about 60 mM and about 100 mM, about 65 mM and about 100 mM, about 70 mM and about 100 mM, about 75 mM and about 100 mM, about 80 mM and about 100 mM, about 85 mM and about 100 mM, about 90 mM and about 100 mM, or about 95 mM and about 100 mM.

[0343] In one embodiment, the concentration of the diafiltration buffer is between about 0.01 mM-50 mM, between about 0.1 mM-50 mM, between about 0.5 mM-50 mM, between about 1 mM-50 mM, between about 2 mM-50 mM, between about 3 mM-50 mM, between about 4 mM-50 mM, between about 5 mM-50 mM, between about 6 mM-50 mM, between about 7 mM-50 mM, between about 8 mM-50 mM, between about 9 mM-50 mM, between about 10 mM-50 mM, between about 11 mM-50 mM, between about 12 mM-50 mM, between about 13 mM-50 mM, between about 14 mM-50 mM, between about 15 mM-50 mM, between about 16 mM-50 mM, between about 17 mM-50 mM, between about 18 mM-50 mM, between about 19 mM-50 mM, between about 20 mM-50 mM, between about 21 mM-50 mM, between about 22 mM-50 mM, between about 24 mM-50 mM, between about 25 mM-50 mM, between about 26 mM-50 mM, between about 27 mM-50 mM, between about 28 mM-50 mM, between about 29 mM-50 mM, between about 30 mM-50 mM, between about 31 mM-50 mM, between about 32 mM-50 mM, between about 34 mM-50 mM, between about 35 mM-50 mM, between about 36 mM-50 mM, between about 37 mM-50 mM, between about 38 mM-5 In some embodiments, the present invention relates to an embodiment of the present invention which is preferably between about 1mM and 50mM, between about 12mM and 50mM, between about 13mM and 50mM, between about 14mM and 50mM, between about 15mM and 50mM, between about 16mM and 50mM, between about 17mM and 50mM, between about 18mM and 50mM, between about 19mM and 50mM, between about 20mM and 50mM, between about 25mM and 50mM, between about 30mM and 50mM, between about 35mM and 50mM, between about 40mM and 50mM, or between about 45mM and 50mM.

[0344] In one embodiment, the concentration of the diafiltration buffer is between about 0.01 mM-25 mM, between about 0.1 mM-25 mM, between about 0.5 mM-25 mM, between about 1 mM-25 mM, between about 2 mM-25 mM, between about 3 mM-25 mM, between about 4 mM-25 mM, between about 5 mM-25 mM, between about 6 mM-25 mM, between about 7 mM-25 mM, between about 8 mM-25 mM, between about 9 mM-25 mM, between about 10 mM-25 mM, between about 11 mM-25 mM, between about 12 mM-25 mM, between about 13 mM-25 mM, between about 14 mM-25 mM, between about 15 mM-25 mM, between about 16 mM-25 mM, between about 17 mM-25 mM, between about 18 mM-25 mM, between about 19 mM-25 mM, between about 20 mM-25 mM, between about 21 mM-25 mM, between about 23 mM-25 mM, between about 24 mM-25 mM, between about 25 mM-25 mM, between about 26 mM-25 mM, between about 27 mM-25 mM, between about 28 mM-25 mM, between about 29 mM-25 mM, between about 30 mM-25 mM, between about 35 mM-25 mM, between about 36 mM-25 mM, between about 37 mM-25 mM, between about 38 mM-25 mM, between about 39 mM-25 mM, between about 40 mM-25 mM, between about 50 mM-2 Between about 9mM-25mM, between about 10mM-25mM, between about 11mM-25mM, between about 12mM-25mM, between about 13mM-25mM, between about 14mM-25mM, between about 15mM-25mM, between about 16mM-25mM, between about 17mM-25mM, between about 18mM-25mM, between about 19mM-25mM or between about 20mM-25mM.

[0345] In one embodiment, the concentration of the diafiltration buffer is between about 0.01 mM-15 mM, between about 0.1 mM-15 mM, between about 0.5 mM-15 mM, between about 1 mM-15 mM, between about 2 mM-15 mM, between about 3 mM-15 mM, between about 4 mM-15 mM, between about 5 mM-15 mM, between about 6 mM-15 mM, between about 7 mM-15 mM, between about 8 mM-15 mM, between about 9 mM-15 mM, between about 10 mM-15 mM, between about 11 mM-15 mM, between about 12 mM-15 mM, between about 13 mM-15 mM, or between about 14 mM-15 mM.

[0346] In one embodiment, the concentration of the diafiltration buffer is between about 0.01mM-10mM, between about 0.1mM-10mM, between about 0.5mM-10mM, between about 1mM-10mM, between about 2mM-10mM, between about 3mM-10mM, between about 4mM-10mM, between about 5mM-10mM, between about 6mM-10mM, between about 7mM-10mM, between about 8mM-10mM, or between about 9mM-10mM.

[0347] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0348] In one embodiment, the concentration of the diafiltration buffer is about 0.01 mM, about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM. or about 100 mM.

[0349] In one embodiment, the concentration of the diafiltration buffer is about 0.1 mM, about 0.2 mM, about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM or about 50 mM. In one embodiment, the concentration of the diafiltration buffer is about 30 mM. In one embodiment, the concentration of the diafiltration buffer is about 25 mM. In one embodiment, the concentration of the diafiltration buffer is about 20 mM. In one embodiment, the concentration of the diafiltration buffer is about 15 mM. In one embodiment, the concentration of the diafiltration buffer is about 10 mM.

[0350] In one embodiment, the diafiltration buffer solution comprises a salt. In some embodiments, the salt is selected from the group consisting of magnesium chloride, potassium chloride, sodium chloride, and combinations thereof. In a specific embodiment, the salt is sodium chloride. In a specific embodiment, the diafiltration buffer solution comprises about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 250, or about 300 mM sodium chloride.

[0351] In an embodiment of the present invention, the number of diafiltration volumes is at least 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50. In one embodiment of the invention, the diafiltration volume number is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95 or about 100. In one embodiment of the present invention, the diafiltration volume number is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14 or about 15.

[0352] In one embodiment of the invention, the ultrafiltration and diafiltration steps are performed at a temperature between about 20° C. and about 90° C. In one embodiment, the ultrafiltration and diafiltration steps are performed at a temperature between about 35° C. and about 80° C., at a temperature between about 40° C. and about 70° C., at a temperature between about 45° C. and about 65° C., at a temperature between about 50° C. and about 60° C., at a temperature between about 50° C. and about 55° C., at a temperature between about 45° C. and about 55° C., or at a temperature between about 45° C. and about 55° C. Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0353] In one embodiment, the ultrafiltration and diafiltration steps are performed at about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C In one embodiment, the ultrafiltration and diafiltration steps are performed at a temperature of about 50° C., about 51° C., about 52° C., about 53° C., about 54° C., about 55° C., about 56° C., about 57° C., about 58° C., about 59° C., about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., about 75° C., about 76° C., about 77° C., about 78° C., about 79° C., or about 80° C. In one embodiment, the ultrafiltration and diafiltration steps are performed at a temperature of about 50° C.

[0354] In one embodiment of the present invention, the diafiltration step is performed at a temperature between about 20° C. and about 90° C. In one embodiment, the diafiltration step is performed at a temperature between about 35° C. and about 80° C., at a temperature between about 40° C. and about 70° C., at a temperature between about 45° C. and about 65° C., at a temperature between about 50° C. and about 60° C., at a temperature between about 50° C. and about 55° C., at a temperature between about 45° C. and about 55° C., or at a temperature between about 45° C. and about 55° C. Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0355] In one embodiment, the diafiltration step is performed at about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C The diafiltration step is performed at a temperature of about 50° C., about 51° C., about 52° C., about 53° C., about 54° C., about 55° C., about 56° C., about 57° C., about 58° C., about 59° C., about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., about 75° C., about 76° C., about 77° C., about 78° C., about 79° C., or about 80° C. In one embodiment, the diafiltration step is performed at a temperature of about 50° C.

[0356] In one embodiment of the present invention, the ultrafiltration step is performed at a temperature between about 20° C. and about 90° C. In one embodiment, the ultrafiltration step is performed at a temperature between about 35° C. and about 80° C., at a temperature between about 40° C. and about 70° C., at a temperature between about 45° C. and about 65° C., at a temperature between about 50° C. and about 60° C., at a temperature between about 50° C. and about 55° C., at a temperature between about 45° C. and about 55° C., or at a temperature between about 45° C. and about 55° C. Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0357] In one embodiment, the ultrafiltration step is performed at about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C The ultrafiltration step is performed at about 80° C., about 50° C., about 51° C., about 52° C., about 53° C., about 54° C., about 55° C., about 56° C., about 57° C., about 58° C., about 59° C., about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., about 75° C., about 76° C., about 77° C., about 78° C., about 79° C., or about 80° C. In one embodiment, the ultrafiltration step is performed at a temperature of about 50° C.

[0358] 1.11 Homogenization / Sizing

[0359] During the purification procedure, the size of the polysaccharide may be slightly reduced.

[0360] In one embodiment, the polysaccharide purified solution of the invention (eg obtained by ultrafiltration and / or diafiltration of fraction 1.10) is not sized.

[0361] In one embodiment, the polysaccharide can be homogenized by a sizing technique. Mechanical or chemical sizing can be used. Chemical hydrolysis can be performed using, for example, acetic acid. Mechanical sizing can be performed using high pressure homogenization shear.

[0362] Therefore, in one embodiment, the polysaccharide purified solution obtained by ultrafiltration and / or diafiltration of fraction 1.10 is sized to a target molecular weight.

[0363] As used herein, the term "molecular weight" of a polysaccharide refers to the molecular weight calculated, for example, by size exclusion chromatography (SEC) coupled with a multi-angle laser light scattering detector (MALLS).

[0364] In some embodiments, the purified polysaccharide size is set to a molecular weight between about 5 kDa and about 4,000 kDa. In other such embodiments, the purified polysaccharide size is set to a molecular weight between about 10 kDa and about 4,000 kDa. In other such embodiments, the purified polysaccharide size is set to a molecular weight between about 50 kDa and about 4,000 kDa. In other such embodiments, the polysaccharide, the purified polysaccharide size is set to a molecular weight between about 50 kDa and about 3,500 kDa, between about 50 kDa and about 3,000 kDa, between about 50 kDa and about 2,500 kDa, between about 50 kDa and about 2,000 kDa, between about 50 kDa and about 1,750 kDa, between about 50 kDa and about 1,500 kDa, between about 50 kDa and about 1,250 kDa, between about 50 kDa and about 1,000 kDa a, between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 100 kDa and about 4,000 kDa, between about 100 kDa and about 3,500 kDa, between about 100 kDa and about 3,000 kDa, between about 100 kDa and about 2,500 kDa, between about 100 kDa and about 2,250 kDa, between about 100 kDa and about 2,000 kDa, between about 100 kDa and about 1,750 kDa kDa, between about 100 kDa and about 1,500 kDa, between about 100 kDa and about 1,250 kDa, between about 100 kDa and about 1,000 kDa, between about 100 kDa and about 750 kDa, between about 100 kDa and about 500 kDa, between about 200 kDa and about 4,000 kDa, between about 200 kDa and about 3,500 kDa, between about 200 kDa and about 3,000 kDa, between about 200 kDa and about kDa, between about 200 kDa and about 1,500 kDa, between about 200 kDa and about 1,250 kDa, between about 200 kDa and about 1,000 kDa, between about 200 kDa and about 750 kDa, or between about 200 kDa and about 500 kDa.In other such embodiments, the polysaccharide, the purified polysaccharide is sized between about 250 kDa and about 3,500 kDa, between about 250 kDa and about 3,000 kDa, between about 250 kDa and about 2,500 kDa, between about 250 kDa and about 2,000 kDa, between about 250 kDa and about 1,750 kDa, between about 250 kDa and about 1,500 kDa, between about 250 kDa and about 1,250 kDa, between about 250 kDa and about 1 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about 500 kDa, between about 300 kDa and about 4,000 kDa, between about 300 kDa and about 3,500 kDa, between about 300 kDa and about 3,000 kDa, between about 300 kDa and about 2,500 kDa, between about 300 kDa and about 2,250 kDa, between about 300 kDa and about 2,000 kDa, between about 300 kDa and about 1,700 kDa, kDa, between about 300 kDa and about 1,500 kDa, between about 300 kDa and about 1,250 kDa, between about 300 kDa and about 1,000 kDa, between about 300 kDa and about 750 kDa, between about 300 kDa and about 500 kDa, between about 500 kDa and about 4,000 kDa, between about 500 kDa and about 3,500 kDa, between about 500 kDa and about 3,000 kDa, between about 500 kDa and about 500 kDa The molecular weight of the peptide may be between about 500 kDa and about 2,500 kDa, between about 500 kDa and about 2,250 kDa, between about 500 kDa and about 2,000 kDa, between about 500 kDa and about 1,750 kDa, between about 500 kDa and about 1,500 kDa, between about 500 kDa and about 1,250 kDa, between about 500 kDa and about 1,000 kDa, between about 500 kDa and about 750 kDa, or between about 500 kDa and about 600 kDa.

[0365] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0366] In some embodiments, the purified polysaccharide is sized to about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 75 kDa, about 90 kDa, about 100 kDa, about 150 kDa, about 200 kDa, about 250 kDa, about 300 kDa, about 350 kDa, about 400 kDa, about 450 kDa, about 500 kDa, about 550 kDa, about 600 kDa, about 650 kDa, about 750 kDa, about 80 ... kDa, about 3000 kDa, about 3250 kDa, about 3500 kDa, about 3750 kDa, about 3000 kDa, about 3250 kDa, about 3500 kDa, about 3750 kDa, or about 4,000 kDa.

[0367] In a preferred embodiment, the purified polysaccharide is a capsular polysaccharide from serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F or 33F of S. pneumoniae, wherein the molecular weight of the capsular polysaccharide is within one of the above ranges or has a size about as described above.

[0368] 1.12 Sterile Filtration

[0369] In one embodiment, the purified polysaccharide solution of the present invention is sterile filtered.

[0370] Thus, in one embodiment, the ultrafiltration and / or diafiltration step of section 1.10 may optionally be followed by a sterile filtration step.

[0371] In one embodiment, if the homogenization / sizing step of section 1.11 is performed, it can optionally be followed by a sterile filtration step.

[0372] In one embodiment, any of the steps in sections 1.2 to 1.9 can optionally be followed by a sterile filtration step.

[0373] In one embodiment, the sterile filtration is dead-end filtration (vertical filtration). In one embodiment, the sterile filtration is tangential filtration.

[0374] In one embodiment, the solution is processed through a sterile filtration step wherein the filter has a nominal retention range of between about 0.01-0.2 microns, about 0.05-0.2 microns, about 0.1-0.2 microns, or about 0.15-0.2 microns.

[0375] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0376] In one embodiment, the solution is processed through a sterile filtration step wherein the filter has a nominal retention range of about 0.05, about 0.1, about 0.15, or about 0.2 microns.

[0377] In one embodiment, the solution is processed through a sterile filtration step wherein the filter has a nominal retention range of about 0.2 microns.

[0378] In one embodiment, the solution is processed through a sterile filtration step, wherein the filter has a flow rate of about 25-1500 L / m 2 、50-1500L / m 2 、75-1500L / m 2 100-1500L / m 2 150-1500L / m 2 、200-1500L / m 2 、250-1500L / m 2 、300-1500L / m 2 、350-1500L / m 2 、400-1500L / m 2 、500-1500L / m 2 、750-1500L / m 2 1000-1500L / m 2 or 1250-1500L / m 2 filtering capacity.

[0379] In one embodiment, the solution is processed through a sterile filtration step, wherein the filter has a flow rate of about 25-1000 L / m 2 、50-1000L / m 2 , 75-1000L / m 2 100-1000L / m 2 150-1000L / m 2 、200-1000L / m 2 、250-1000L / m 2 、300-1000L / m 2 、350-1000L / m 2 、400-1000L / m 2 、500-1000L / m 2 or 750-1000L / m 2 filtering capacity.

[0380] In one embodiment, the solution is processed through a sterile filtration step, wherein the filter has a filtration capacity of 25-500 L / m 2 , 50-500L / m 2 、75-500L / m 2 100-500L / m 2 150-500L / m 2 、200-500L / m 2 、250-500L / m 2 、300-500L / m 2 、350-500L / m 2 or 400-500L / m 2 filtering capacity.

[0381] In one embodiment, the solution is processed through a sterile filtration step, wherein the filter has a 25-300 L / m 2 、50-300L / m 2 , 75-300L / m 2 100-300L / m 2 、150-300L / m 2 , 200-300L / m 2 or 250-300L / m 2 filtering capacity.

[0382] In one embodiment, the solution is processed through a sterile filtration step, wherein the filter has a 25-250 L / m 2 、50-250L / m 2 、75-250L / m 2 100-250L / m 2 or 150-250L / m 2 , 200-250L / m 2 filtering capacity.

[0383] In one embodiment, the solution is processed through a sterile filtration step, wherein the filter has a 25-100 L / m 2 , 50-100L / m 2 or 75-100L / m 2 filtering capacity.

[0384] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0385] In one embodiment, the solution is processed by a microfiltration step, wherein the filter has a flow rate of about 25, about 50, about 75, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, or about 1500 L / m 2 filtering capacity.

[0386] 1.13 Final Materials

[0387] The polysaccharide can be made into a liquid solution. The polysaccharide can be further processed (e.g. freeze-dried into a dry powder, see WO2006 / 110381). Therefore, in one embodiment, the polysaccharide is a dry powder.

[0388] In one embodiment, the polysaccharide is in the form of a lyophilized cake.

[0389] 2. Uses of purified polysaccharides

[0390] The polysaccharide purified by the method of the present invention can be used as an antigen. The pure polysaccharide is used as an antigen in a vaccine (see 23-valent non-conjugated pneumococcal polysaccharide vaccine PNEUMOVAX).

[0391] The polysaccharides purified by the method of the present invention can also be conjugated to a carrier protein to obtain a glycoconjugate.

[0392] 2.1. Glycoconjugates

[0393] The polysaccharide purified by the method of the present invention can be conjugated to a carrier protein to obtain a glycoconjugate.

[0394] For the purposes of the present invention, the term 'glycoconjugate' refers to a saccharide covalently linked to a carrier protein. In one embodiment the saccharide is directly linked to the carrier protein. In a second embodiment the saccharide is linked to the carrier protein via a spacer / linker group.

[0395] In general, covalent attachment of a saccharide to a carrier enhances the immunogenicity of the saccharide because it converts the saccharide from a T-independent antigen to a T-dependent antigen, thereby eliciting immunological memory. Conjugation is particularly useful for pediatric vaccines.

[0396] Polysaccharides purified by the methods of the invention may be activated (eg, chemically activated) to enable them to react (eg, with a linking group or directly with a carrier protein), and subsequently incorporated into a glycoconjugate, as further described herein.

[0397] The purified polysaccharide size can be adjusted to a target molecular weight before conjugation, for example by the method disclosed in Section 1.11 above. Therefore, in one embodiment, the purified polysaccharide is sized before conjugation. In one embodiment, the purified polysaccharide as disclosed herein can be sized before conjugation to obtain oligosaccharides. Oligosaccharides have a low number of repeating units (usually 5-15 repeating units) and are typically derived by sizing the polysaccharide (e.g., hydrolysis).

[0398] However, preferably, the carbohydrate used for conjugation is a polysaccharide. High molecular weight polysaccharides are able to induce certain antibody immune responses due to the epitopes present on the surface of the antigen. It is preferred to encompass the isolation and purification of high molecular weight polysaccharides for use in the conjugates of the present invention.

[0399] Thus, in one embodiment, the polysaccharide is sized and remains a polysaccharide.

[0400] In one embodiment, the polysaccharide is not sized.

[0401] In some embodiments, the purified polysaccharide prior to conjugation (after sizing or without sizing) has a molecular weight between 5 kDa and 4,000 kDa. In other such embodiments, the purified polysaccharide has a molecular weight between 10 kDa and 4,000 kDa. In other such embodiments, the purified polysaccharide has a molecular weight between 50 kDa and 4,000 kDa. In other such embodiments, the purified polysaccharide has a molecular weight between 50 kDa and 3,500 kDa, between 50 kDa and 3,000 kDa, between 50 kDa and 2,500 kDa, between 50 kDa and 2,000 kDa, between 50 kDa and 1,750 kDa, between 50 kDa and 1,500 kDa, between 50 kDa and 1,250 kDa, between 50 kDa and 1,0 ...2,500 kDa, between 50 kDa and 2,000 kDa, between 50 kDa and 1,750 kDa, between 50 kDa and 1,500 kDa, between 50 kDa and 1,250 kDa, between 50 kDa and 1,000 kDa, between 50 kDa and 2,500 kDa, between 50 kDa and 2,000 kDa, between 50 kDa and 1,500 kDa, between 50 kDa and 1,250 kDa, between 50 kDa and 1,000 kDa, between 50 kDa and 2,500 kDa, between 50 kDa and 2,500 kDa, between 50 kDa and 750 kDa, between 50 kDa and 500 kDa, between 100 kDa and 4,000 kDa, between 100 kDa and 3,500 kDa, between 100 kDa and 3,000 kDa, between 100 kDa and 2,500 kDa, between 100 kDa and 2,250 kDa, between 100 kDa and 2,000 kDa, between 100 kDa and 1,75 ...500 kDa, between 100 kDa and 2,250 kDa, between 100 kDa and 2,000 kDa, between 100 kDa and 1,750 kDa, between 100 kDa and 2,500 kDa, between 100 kDa and 2,500 kDa, between 100 kDa and 2 between 100 kDa and 1,500 kDa, between 100 kDa and 1,250 kDa, between 100 kDa and 1,000 kDa, between 100 kDa and 750 kDa, between 100 kDa and 500 kDa, between 200 kDa and 4,000 kDa, between 200 kDa and 3,500 kDa, between 200 kDa and 3,000 kDa, between 200 kDa and 2,5 kDa, between 200 kDa and 2,250 kDa, between 200 kDa and 2,000 kDa, between 200 kDa and 1,750 kDa, between 200 kDa and 1,500 kDa, between 200 kDa and 1,250 kDa, between 200 kDa and 1,000 kDa, between 200 kDa and 750 kDa, or between 200 kDa and 500 kDa.In other such embodiments, the polysaccharide has a molecular weight of between 250 kDa and 3,500 kDa, between 250 kDa and 3,000 kDa, between 250 kDa and 2,500 kDa, between 250 kDa and 2,000 kDa, between 250 kDa and 1,750 kDa, between 250 kDa and 1,500 kDa, between 250 kDa and 1,250 kDa, between 250 kDa and 1,000 kDa. a, between 250 kDa and 750 kDa, between 250 kDa and 500 kDa, between 300 kDa and 4,000 kDa, between 300 kDa and 3,500 kDa, between 300 kDa and 3,000 kDa, between 300 kDa and 2,500 kDa, between 300 kDa and 2,250 kDa, between 300 kDa and 2,000 kDa, between 300 kDa and 1,750 kDa kDa, between 300 kDa and 1,500 kDa, between 300 kDa and 1,250 kDa, between 300 kDa and 1,000 kDa, between 300 kDa and 750 kDa, between 300 kDa and 500 kDa, between 500 kDa and 4,000 kDa, between 500 kDa and 3,500 kDa, between 500 kDa and 3,000 kDa, between 500 kDa and 2 The molecular weight of the present invention is between 500 kDa and 2,250 kDa, between 500 kDa and 2,000 kDa, between 500 kDa and 1,750 kDa, between 500 kDa and 1,500 kDa, between 500 kDa and 1,250 kDa, between 500 kDa and 1,000 kDa, between 500 kDa and 750 kDa, or between 500 kDa and 600 kDa. Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0402] In some embodiments, the purified polysaccharide has a molecular weight of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 75 kDa, 90 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, kDa, 3000 kDa, 3250 kDa, 3500 kDa, 3750 kDa, or 4,000 kDa.

[0403] In one embodiment, the purified polysaccharide is a capsular saccharide (polysaccharide or oligosaccharide).

[0404] In one embodiment, the purified polysaccharide is a capsular polysaccharide from E. coli. In one embodiment, the purified polysaccharide is a capsular polysaccharide from an E. coli portion of the diarrheagenic E. coli group (EEC group), such as enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), O157:H7 enterohemorrhagic E. coli (EHEC), or enteroinvasive E. coli (EIEC). In one embodiment, the purified polysaccharide is a capsular polysaccharide from uropathogenic E. coli (UPEC).

[0405] In one embodiment, the purified polysaccharide is a capsular polysaccharide from an E. coli serotype selected from the group consisting of serotypes O157:H7, O26:H11, O111:H-, and O103:H2. In one embodiment, the purified polysaccharide is a capsular polysaccharide from an E. coli serotype selected from the group consisting of serotypes O6:K2:H1 and O18:K1:H7. In one embodiment, the purified polysaccharide is a capsular polysaccharide from an E. coli serotype selected from the group consisting of serotypes O45:K1, O17:K52:H18, O19:H34, and O7:K1. In one embodiment, the purified polysaccharide is a capsular polysaccharide from an E. coli serotype O104:H4. In one embodiment, the purified polysaccharide is a capsular polysaccharide from an E. coli serotype O1:K12:H7. In one embodiment, the purified polysaccharide is a capsular polysaccharide from an E. coli serotype O127:H6. In one embodiment, the purified polysaccharide is a capsular polysaccharide from E. coli serotype O139:H28. In one embodiment, the purified polysaccharide is a capsular polysaccharide from E. coli serotype O128:H2.

[0406] In another embodiment, the purified polysaccharide is a capsular polysaccharide from Neisseria meningitidis. In one embodiment, the purified polysaccharide is a capsular polysaccharide from Neisseria meningitidis serogroup A (MenA), Neisseria meningitidis serogroup W135 (MenW135), Neisseria meningitidis serogroup Y (MenY), Neisseria meningitidis serogroup X (MenX) or Neisseria meningitidis serogroup C (MenC).

[0407] In another embodiment, the purified polysaccharide is a capsular polysaccharide from Klebsiella pneumoniae. In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O1 (O1), Klebsiella pneumoniae serogroup O2 (O2), Klebsiella pneumoniae serogroup O2ac (O2ac), Klebsiella pneumoniae serogroup O3 (O3), Klebsiella pneumoniae serogroup O4 (O4), Klebsiella pneumoniae serogroup O5 (O5), Klebsiella pneumoniae serogroup O7 (O7), Klebsiella pneumoniae serogroup O8 (O8) or Klebsiella pneumoniae serogroup O9 (O9). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O1 (O1). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O2 (O2). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O2ac (O2ac). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O3 (O3). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O4 (O4). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O5 (O5). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O7 (O7). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O8 (O8). In one embodiment, the source of the bacterial capsular polysaccharide is Klebsiella pneumoniae serogroup O9 (O9).

[0408] Any suitable conjugation reaction may be used, using any suitable linking group as necessary. See, for example, WO2007116028, pages 17-22.

[0409] The purified oligosaccharides or polysaccharides described herein are chemically activated to render the saccharides capable of reacting with a carrier protein.

[0410] In one embodiment, the glycoconjugate is prepared using reductive amination.

[0411] Reductive amination involves two steps: (1) oxidation (activation) of the purified carbohydrate and (2) the activation of the carbohydrate and a carrier protein (e.g., CRM). 197 , DT, TT or PD) to form a glycoconjugate (see, for example, WO2015110941, WO2015110940).

[0412] As mentioned above, prior to oxidation, the size of the polysaccharide can be set to a target molecular weight (MW) range. Mechanical or chemical hydrolysis can be used. Chemical hydrolysis can be performed using acetic acid. In one embodiment, the size of the purified polysaccharide is reduced by mechanical homogenization.

[0413] In one embodiment, the purified polysaccharide or oligosaccharide is conjugated to a carrier protein by a process comprising the following steps:

[0414] (a) reacting the purified polysaccharide or oligosaccharide with an oxidizing agent;

[0415] (b) optionally quenching the oxidation reaction by adding a quencher;

[0416] (c) complexing the activated polysaccharide or oligosaccharide of step (a) or (b) with a carrier protein; and

[0417] (d) reacting the complexed activated polysaccharide or oligosaccharide and carrier protein with a reducing agent to form a glycoconjugate.

[0418] After the oxidation step (a), the sugars are said to be activated and are referred to as "activated polysaccharides or oligosaccharides".

[0419] The oxidation step (a) may involve a reaction with a periodate. For the purposes of the present invention, the term "periodate" includes periodate and periodic acid; the term also includes metaperiodate (I 4 - ) and orthoperiodate (IO 6 5- ) and various periodates (e.g. sodium periodate and potassium periodate).

[0420] In a preferred embodiment, the oxidizing agent is sodium periodate. In one embodiment, the periodate used for oxidation is metaperiodate. In one embodiment, the periodate used for oxidation is sodium metaperiodate.

[0421] The oxidation step (a) may involve reacting with a stable nitroxyl or nitroxy radical compound such as piperidine-N-oxy or pyrrolidine-N-oxy compound in the presence of an oxidant to selectively oxidize the primary hydroxyl groups of the polysaccharide or oligosaccharide to produce activated saccharides containing aldehyde groups (see WO2014097099). In one aspect, the stable nitroxyl or nitroxy radical compound is any one disclosed in WO2014097099, page 3, line 14 to page 4, line 7, and the oxidant is any one disclosed in WO2014097099, page 4, lines 8 to 15. In one aspect, the stable nitroxyl or nitroxy radical compound is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and the oxidant is N-chlorosuccinimide (NCS).

[0422] In one embodiment, the quencher is as disclosed in WO2015110941 (see page 30, lines 3 to 26).

[0423] In one embodiment, the reduction reaction (d) is carried out in an aqueous solvent. In one embodiment, the reduction reaction (d) is carried out in an aprotic solvent. In one embodiment, the reduction reaction (d) is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent.

[0424] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride or zinc borohydride in the presence of a Bronsted acid or a Lewis acid, an amine borane such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe i PrN-BH 3 , benzylamine-BH 3 or 5-ethyl-2-methylpyridine borane (PEMB). In a preferred embodiment, the reducing agent is sodium cyanoborohydride.

[0425] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate, which can be capped using a suitable capping agent. In one embodiment, this capping agent is sodium borohydride (NaBH 4 ).

[0426] After conjugation to a carrier protein, the glycoconjugate can be purified (enriched in terms of the amount of glyco-protein conjugate) by a variety of techniques known to the skilled person. These techniques include dialysis, concentration / diafiltration procedures, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography) and depth filtration.

[0427] In one embodiment, the glycoconjugates are prepared using cyanidation chemistry.

[0428] In one embodiment, the purified polysaccharide or oligosaccharide is activated with cyanogen bromide. The activation corresponds to cyanation of the hydroxyl groups of the polysaccharide or oligosaccharide. The activated polysaccharide or oligosaccharide is then coupled directly or via a spacer (linker) group to an amino group on a carrier protein.

[0429] In one embodiment, the purified polysaccharide or oligosaccharide is activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide or oligosaccharide is then coupled to the amino group on the carrier protein directly or through a spacer (linker) group.

[0430] In one embodiment, the spacer group can be cystamine or cysteamine to produce a thiolated polysaccharide or oligosaccharide, which can be coupled to the carrier via a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyryloxy]succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g., using iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl) aminobenzoate (SlAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA) or succinimidyl 3-[bromoacetylamino] propionate (SBAP)). Preferably, the cyanate ester (optionally prepared by CDAP chemistry) is coupled with hexamethylenediamine or adipic acid dihydrazide (ADH), and the amino-derivatized sugar is attached to the carrier protein (e.g., CRM) via the carboxyl groups on the protein carrier using carbodiimide (e.g., EDAC or EDC) chemistry. 197 Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348 and WO 96 / 129094.

[0431] In one embodiment, the sugar conjugate is prepared by using a di-electrophile such as carbonyldiimidazole (CDI) or carbonylditriazole (CDT). In such embodiments, the conjugation reaction is preferably carried out in an aprotic solvent such as DMF or DMSO by a direct route or using a bigeneric linker (see, e.g., WO2011041003).

[0432] In one embodiment, the glycoconjugate is prepared by a method for preparing a glycoconjugate as disclosed in WO2014027302. The resulting glycoconjugate comprises a saccharide covalently conjugated to a carrier protein via a divalent heterobifunctional spacer group (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC). Alternatively, the glycoconjugate is prepared by a method for preparing a glycoconjugate as disclosed in WO2015121783.

[0433] Other suitable conjugation techniques use carbodiimides such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDC plus sulfonyl NHS, CMC (1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide), DCC (N,N'-dicyclohexylcarbodiimide) or DIC (diisopropylcarbodiimide).

[0434] In one embodiment, the polysaccharide or oligosaccharide is conjugated to the carrier protein via a linking group, such as a bifunctional linking group. The linking group is optionally heterobifunctional or homobifunctional, having, for example, a reactive amino group and a reactive carboxylic acid group, 2 reactive amino groups or two reactive carboxylic acid groups. The linking group has, for example, between 4 and 20, between 4 and 12, between 5 and 10 carbon atoms. A possible linking group is adipic acid dihydrazide (ADH). Other linking groups include B-propionylamino (WO00 / 10599), nitrophenylethylamine, haloalkyl halides), glycosidic bonds (US4673574, US4808700), hexamethylenediamine and 6-aminocaproic acid (US4459286).

[0435] Carrier Protein

[0436] The components of the glycoconjugate are carrier proteins conjugated to purified polysaccharides or oligosaccharides. The terms "protein carrier" or "carrier protein" or "carrier" are used interchangeably herein. The carrier protein should be suitable for standard conjugation procedures.

[0437] In a preferred embodiment, the carrier protein of the glycoconjugate is selected from the group consisting of: DT (diphtheria toxin), TT (tetanus toxoid) or fragment C of TT, CRM 197 (a nontoxic but antigenically identical diphtheria toxin variant), other DT mutations (such as CRM 176 、CRM 228 、CRM 45 (Uchida et al. (1973) J. Biol. Chem. 218: 3838-3844), CRM 9 、CRM 102 、CRM 103 or CRM 107and other mutations described in Nicholls and Youle, Genetically Engineered Toxins, ed. Frankel, Maecel Dekker Inc. (1992); deletion or mutation of Glu-148 to Asp, Gln or Ser and / or deletion or mutation of Ala 158 to Gly and other mutations disclosed in U.S. Pat. Nos. 4,709,017 and 4,950,740; mutation of at least one or more residues Lys 516, Lys 526, Phe 530 and / or Lys 534 and other mutations disclosed in U.S. Pat. Nos. 5,917,017 and 6,455,673; or fragments disclosed in U.S. Pat. No. 5,843,711, pneumococcal hemolysin (ply) (Kuo et al. (1995) Infect Immunity Immun) 63: 2706-2713) comprises ply that is detoxified in some way, such as dPLY-GMBS (WO 2004 / 081515, WO 2006 / 032499) or dPLY-formaldehyde, PhtX, comprises PhtA, PhtB, PhtD, PhtE (the sequences of PhtA, PhtB, PhtD or PhtE are disclosed in WO 00 / 37105 and WO 00 / 39299) fusions of Pht proteins, such as PhtDE fusions, PhtBE fusions, Pht AE (WO 01 / 98334, WO03 / 054007, WO 2009 / 000826), OMPC (meningococcal outer membrane protein), which is usually extracted from Neisseria meningitidis serogroup B (EP0372501), PorB (from Neisseria meningitidis), PD (Haemophilus influenzae protein D;See, for example, EP0594610 B), or immunologically functional equivalents thereof, synthetic peptides (EP0378881, EP0427347), heat shock proteins (WO 93 / 17712, WO 94 / 03208), pertussis proteins (WO 98 / 58668, EP0471177), cytokines, lymphokines, growth factors or hormones (WO 91 / 01146), artificial proteins including a variety of human CD4+T cell epitopes from various pathogen-derived antigens (Falugi et al. (2001) Eur J Immunol 31:3816-3824), such as N19 protein (Baraldoi et al. (2004) Infect Immunology 72:4884-4887), pneumococcal surface protein PspA (WO 02 / 091998), iron uptake proteins (WO 01 / 72337), Clostridium difficile toxin A or B (WO 00 / 61761), transferrin binding protein, pneumococcal adhesion protein (PsaA), recombinant Pseudomonas aeruginosa exotoxin A (especially non-toxic mutations thereof (e.g., exotoxin A having a substitution at glutamic acid 553 (Douglas et al. (1987) J. Bacteriol. 169(11):4967-4971)). Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD) can also be used as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins, such as cholera toxoid (e.g., such as WO 2004 / 083251), E. coli LT, E. coli ST and exotoxin A from Pseudomonas aeruginosa.;

[0438] In a preferred embodiment, the carrier protein of the glycoconjugate is independently selected from the group consisting of: TT, DT, DT mutation (such as CRM 197 ), Haemophilus influenzae protein D, PhtX, PhtD, PhtDE fusions (particularly those described in WO 01 / 98334 and WO03 / 054007), detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, toxin A or B of Clostridium difficile and PsaA.

[0439] In one embodiment, the carrier protein of the glycoconjugate is DT (diphtheria toxoid). In another embodiment, the carrier protein of the glycoconjugate is TT (tetanus toxoid).

[0440] In another embodiment, the carrier protein of the glycoconjugate is PD (Haemophilus influenzae protein D; see, eg, EP0594610B).

[0441] In a preferred embodiment, the purified polysaccharide or oligosaccharide is 197 Protein conjugation. CRM 197 The protein is a nontoxic form of diphtheria toxin but is immunologically indistinguishable from diphtheria toxin. CRM 197 Non-toxigenic phage β197 tox- Produced by Corynebacterium diphtheriae infected with the bacteriophage, the phage was generated by nitrosoguanidine mutagenesis of toxigenic coryneform phage β (Uchida et al. (1971) Nature New Biology 233:8-11). CRM 197 The protein has the same molecular weight as diphtheria toxin, but differs in a single base change in its structural gene (guanine to adenine). This single base change results in an amino acid substitution in the mature protein (glutamic acid to glycine) and eliminates the toxicity of diphtheria toxin. CRM 197 Protein is a safe and effective T cell-dependent carbohydrate carrier. About CRM 197 Further details of the invention and its generation can be found, for example, in US Pat. No. 5,614,382.

[0442] In one embodiment, the purified polysaccharide or oligosaccharide is mixed with CRM 197 Protein or CRM 197 In one embodiment, the purified polysaccharide or oligosaccharide is conjugated with a CRM obtained by expressing a recombinant E. coli. 197 A chain conjugation (see CN103495161).

[0443] Preferably, the ratio of carrier protein to polysaccharide or oligosaccharide in the glycoconjugate is between 1:5 and 5:1; for example, between 1:0.5 and 4:1, between 1:1 and 3.5:1, between 1.2:1 and 3:1, between 1.5:1 and 2.5:1; for example, between 1:2 and 2.5:1 or between 1:1 and 2:1 (w / w). In one embodiment, the ratio of carrier protein to polysaccharide or oligosaccharide in the glycoconjugate is about 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1 or 1.6:1.

[0444] After conjugation to a carrier protein, the glycoconjugate can be purified (enriched in terms of the amount of glyco-protein conjugate) by a variety of techniques known to the skilled person. These techniques include dialysis, concentration / diafiltration procedures, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography) and depth filtration.

[0445] The composition may contain a small amount of free carrier. When a given carrier protein is present in free and conjugated form in the composition of the invention, the unconjugated form preferably does not exceed 5% of the total amount of carrier protein in the entire composition, and more preferably is present at less than 2% by weight.

[0446] 2.2 Immunogenic Compositions

[0447] In one embodiment, the invention relates to an immunogenic composition comprising any purified polysaccharide and / or glycoconjugate disclosed herein.

[0448] In one embodiment, the invention relates to an immunogenic composition comprising any of the glycoconjugates disclosed herein.

[0449] In one embodiment, the invention relates to an immunogenic composition comprising 1 to 25 different glycoconjugates disclosed in Section 2.1.

[0450] In one embodiment, the present invention relates to an immunogenic composition comprising 1 to 25 saccharide conjugates from different serotypes of Streptococcus pneumoniae (1 to 25 pneumococcal conjugates). In one embodiment, the present invention relates to an immunogenic composition comprising saccharide conjugates from 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 different serotypes of Streptococcus pneumoniae. In one embodiment, the immunogenic composition comprises saccharide conjugates from 16 or 20 different serotypes of Streptococcus pneumoniae. In one embodiment, the immunogenic composition is a 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 14, 15, 16, 17, 18 or 19 valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 16-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 19-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 20-valent pneumococcal conjugate composition.

[0451] In one embodiment, the immunogenic composition includes saccharide conjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F.

[0452] In one embodiment, the immunogenic composition further comprises saccharide conjugates from S. pneumoniae serotypes 1, 5 and 7F.

[0453] In one embodiment, any of the above immunogenic compositions further comprises saccharide conjugates from S. pneumoniae serotypes 6A and 19A.

[0454] In one embodiment, any of the above immunogenic compositions further comprises a saccharide conjugate from S. pneumoniae serotype 3.

[0455] In one embodiment, any of the above immunogenic compositions further comprises saccharide conjugates from S. pneumoniae serotypes 22F and 33F.

[0456] In one embodiment, any of the above immunogenic compositions further comprises a saccharide conjugate from S. pneumoniae serotypes 8, 10A, 11A, 12F and 15B.

[0457] In one embodiment, any of the above immunogenic compositions further comprises a saccharide conjugate from S. pneumoniae serotype 2.

[0458] In one embodiment, any of the above immunogenic compositions further comprises a saccharide conjugate from S. pneumoniae serotype 9N.

[0459] In one embodiment, any of the above immunogenic compositions further comprises a saccharide conjugate from S. pneumoniae serotype 17F.

[0460] In one embodiment, any of the above immunogenic compositions further comprises a saccharide conjugate from S. pneumoniae serotype 20.

[0461] In one embodiment, the immunogenic composition of the invention comprises saccharide conjugates from S. pneumoniae serotypes 8, 10A, 11A, 12F, 15B, 22F and 33F.

[0462] In one embodiment, any of the above immunogenic compositions further comprises a saccharide conjugate from S. pneumoniae serotype 2.

[0463] In one embodiment, any of the above immunogenic compositions further comprises a saccharide conjugate from S. pneumoniae serotype 9N.

[0464] In one embodiment, any of the above immunogenic compositions further comprises a saccharide conjugate from S. pneumoniae serotype 17F.

[0465] In one embodiment, any of the above immunogenic compositions further comprises a saccharide conjugate from S. pneumoniae serotype 20.

[0466] However, in a preferred embodiment, each saccharide is conjugated individually to a different molecule of a protein carrier (each molecule of the protein carrier has only one type of saccharide conjugated to it). In said embodiment, the capsular saccharide is said to be conjugated individually to the carrier protein. Preferably, all saccharide conjugates of the above immunogenic composition are conjugated individually to the carrier protein.

[0467] In one embodiment of any of the above immunogenic compositions, the saccharide conjugate from S. pneumoniae serotype 22F is combined with CRM 197 In one embodiment of any of the above immunogenic compositions, the saccharide conjugate from S. pneumoniae serotype 33F is conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, a saccharide conjugate from S. pneumoniae serotype 15B is conjugated to CRM197. In one embodiment of any of the above immunogenic compositions, a saccharide conjugate from S. pneumoniae serotype 12F is conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, a saccharide conjugate from S. pneumoniae serotype 10A is conjugated to CRM197. In one embodiment of any of the above immunogenic compositions, a saccharide conjugate from S. pneumoniae serotype 11A is conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, the saccharide conjugate from S. pneumoniae serotype 8 is conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, saccharide conjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F are conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, the saccharide conjugates from S. pneumoniae serotypes 1, 5 and 7F are conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, the saccharide conjugates from S. pneumoniae serotypes 6A and 19A are conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, the saccharide conjugate from S. pneumoniae serotype 3 is conjugated to CRM 197 Conjugation.

[0468] In one embodiment, all of the glycoconjugates of any of the above immunogenic compositions are individually combined with CRM 197 Conjugation.

[0469] In one embodiment, a saccharide conjugate from S. pneumoniae serotype 1, 4, 5, 6B, 7F, 9V, 14 and / or 23F of any of the above immunogenic compositions is conjugated alone to PD.

[0470] In one embodiment, the saccharide conjugate from S. pneumoniae serotype 18C of any of the above immunogenic compositions is conjugated to TT.

[0471] In one embodiment, the saccharide conjugate from S. pneumoniae serotype 19F of any of the above immunogenic compositions is conjugated to DT.

[0472] In one embodiment, the saccharide conjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and / or 23F of any of the above immunogenic compositions are conjugated to PD alone, the saccharide conjugates from S. pneumoniae serotype 18C are conjugated to TT, and the saccharide conjugates from S. pneumoniae serotype 19F are conjugated to DT.

[0473] In one embodiment, the immunogenic composition comprises 8 to 20 different serotypes of S. pneumoniae.

[0474] In one embodiment, the present invention relates to an immunogenic composition comprising 1 to 5 saccharide conjugates from different Neisseria meningitidis serogroups (1 to 5 meningococcal conjugates). In one embodiment, the present invention relates to an immunogenic composition comprising saccharide conjugates from 1, 2, 3, 4 or 5 different Neisseria meningitidis serogroups. In one embodiment, the immunogenic composition comprises 4 or 5 different Neisseria meningitidis. In one embodiment, the immunogenic composition is a 1, 2, 3, 4 or 5-valent meningococcal conjugate composition. In one embodiment, the immunogenic composition is a 2-valent meningococcal conjugate composition. In one embodiment, the immunogenic composition is a 4-valent meningococcal conjugate composition. In one embodiment, the immunogenic composition is a 5-valent meningococcal conjugate composition.

[0475] In one embodiment, the immunogenic composition includes conjugated Neisseria meningitidis serogroup Y capsular saccharide (MenY) and / or conjugated Neisseria meningitidis serogroup C capsular saccharide (MenC).

[0476] In one embodiment, the immunogenic composition includes conjugated Neisseria meningitidis serogroup A capsular saccharide (MenA), conjugated Neisseria meningitidis serogroup W135 capsular saccharide (MenW135), conjugated Neisseria meningitidis serogroup Y capsular saccharide (MenY) and / or conjugated Neisseria meningitidis serogroup C capsular saccharide (MenC).

[0477] In one embodiment, the immunogenic composition includes conjugated Neisseria meningitidis serogroup W135 capsular saccharides (MenW135), conjugated Neisseria meningitidis serogroup Y capsular saccharides (MenY) and / or conjugated Neisseria meningitidis serogroup C capsular saccharides (MenC).

[0478] In one embodiment, the immunogenic composition includes conjugated Neisseria meningitidis serogroup A capsular saccharide (MenA), conjugated Neisseria meningitidis serogroup W135 capsular saccharide (MenW135), conjugated Neisseria meningitidis serogroup Y capsular saccharide (MenY), conjugated Neisseria meningitidis serogroup C capsular saccharide (MenC) and / or conjugated Neisseria meningitidis serogroup X capsular saccharide (MenX).

[0479] In some embodiments, the immunogenic compositions disclosed herein may further include at least one, two or three adjuvants. In some embodiments, the immunogenic compositions disclosed herein may further include an adjuvant. The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An antigen may be primarily used as a delivery system, primarily as an immunomodulator, or have powerful features of both. Suitable adjuvants include those suitable for use in mammals including humans.

[0480] Examples of known suitable delivery system-type adjuvants that can be used in humans include, but are not limited to, alum (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide), calcium phosphate, liposomes, oil-in-water emulsions such as MF59 (4.3% w / v squalene, 0.5% w / v polysorbate 80 (Tween 80), 0.5% w / v sorbitan trioleate (Span 85)), water-in-oil emulsions such as Montanide, and poly(D,L-lactide-co-glycolide) (PLG) microparticles or nanoparticles.

[0481] In one embodiment, the immunogenic composition disclosed herein includes an aluminum salt (alum) as an adjuvant (e.g., aluminum phosphate, aluminum sulfate or aluminum hydroxide). In a preferred embodiment, the immunogenic composition disclosed herein includes aluminum phosphate or aluminum hydroxide as an adjuvant.

[0482] Other exemplary adjuvants that enhance the effectiveness of the immunogenic compositions disclosed herein include, but are not limited to: (1) oil-in-water emulsion formulations (with or without other specific immunostimulants, such as muramyl peptides (see below) or bacterial cell wall components), such as (a) SAF, containing 10% squalane, 0.4% Tween 80, 5% Pluronic block polymer L121 and thr-MDP, microfluidized into a submicron emulsion or vortexed to produce an emulsion with larger particle size, and (b) RIBI TM Adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT), containing 2% squalene, 0.2% Tween 80 and one or more bacterial cell wall components, such as monophosphoryl lipid A (MPL), trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferably MPL + CWS (DETOXTM ); (2) saponin adjuvants, such as QS21, STIMULON TM (Cambridge Bioscience, Worcester, MA), (Isconova, Sweden) or (Commonwealth Serum Laboratories, Australia), or particles produced therefrom, such as ISCOMs (immunostimulatory complexes), wherein the ISCOMS may not contain additional detergents (e.g., WO 00 / 07621); (3) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (4) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (e.g., WO 99 / 44636)), interferons (e.g., interferon-γ), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) (see, e.g., GB-2220221, EP0689454), when used with pneumococcal saccharides, optionally in the substantial absence of alum (see, e.g., WO 00 / 56358); (6) 3dMPL in combination with, e.g., QS21 and / or an oil-in-water emulsion (see, e.g., EP0835318, EP0735898, EP0761231); (7) polyoxyethylene ethers or polyoxyethylene esters (see, e.g., WO 99 / 52549); (8) polyoxyethylene sorbitan ester surfactants in combination with octoxynol (see, e.g., WO 01 / 21207) or a combination of a polyoxyethylene alkyl ether or ester surfactant with at least one additional nonionic surfactant such as octoxynol (e.g., WO 01 / 21152); (9) saponin and immunostimulatory oligonucleotides (e.g., CpG oligonucleotides) (e.g., WO 00 / 62800); (10) immunostimulants and metal salt particles (see, e.g., WO 00 / 23105); (11) saponin and oil-in-water emulsions (e.g., WO 99 / 11241); (12) saponin (e.g., QS21) + 3dMPL + IM2 (optionally + sterol) (e.g., WO 98 / 57659); (13) other substances that act as immunostimulants to enhance the efficacy of the composition. Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamylamino-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE), etc.

[0483] In one embodiment of the present invention, the immunogenic composition as disclosed herein comprises a CpG oligonucleotide as an adjuvant.

[0484] The immunogenic compositions can be formulated in liquid form (i.e., solutions or suspensions) or in lyophilized form. Liquid formulations can advantageously be administered directly from their packaging form, and are therefore ideal for injection, without the need for reconstitution in an aqueous medium, as is otherwise required for lyophilized compositions of the invention.

[0485] The preparation of the immunogenic compositions of the present disclosure can be accomplished using methods recognized in the art. For example, individual polysaccharides and / or conjugates can be formulated with a physiologically acceptable vehicle to prepare a composition. Examples of such vehicles include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and glucose solutions.

[0486] The present disclosure provides an immunogenic composition comprising any combination of polysaccharides or glycoconjugates disclosed herein and a pharmaceutically acceptable excipient, carrier or diluent.

[0487] In one embodiment, the immunogenic composition of the present disclosure is in liquid form, preferably in aqueous liquid form.

[0488] The immunogenic compositions of the present disclosure may include one or more of the following: a buffer, a salt, a divalent cation, a non-ionic detergent, a cryoprotectant such as a sugar, and an antioxidant such as a free radical scavenger or chelator, or any combination thereof.

[0489] In one embodiment, the immunogenic composition of the present disclosure includes a buffer. In one embodiment, the buffer has a pKa of about 3.5 to about 7.5. In some embodiments, the buffer is phosphate, succinate, histidine or citrate. In certain embodiments, the buffer is succinate at a final concentration of 1 mM to 10 mM. In a specific embodiment, the final concentration of the succinate buffer is about 5 mM.

[0490] In one embodiment, the immunogenic composition of the present disclosure includes a salt. In some embodiments, the salt is selected from the group consisting of magnesium chloride, potassium chloride, sodium chloride, and combinations thereof. In a specific embodiment, the salt is sodium chloride. In a specific embodiment, the immunogenic composition of the present invention includes 150 mM sodium chloride.

[0491] In one embodiment, the immunogenic composition of the present disclosure includes a surfactant. In one embodiment, the surfactant is selected from the group consisting of: polysorbate 20 (TWEEN TM 20) Polysorbate 40 (TWEEN TM 40), polysorbate 60 (TWEEN TM 60), polysorbate 65 (TWEEN TM 65) Polysorbate 80 (TWEEN TM80), polysorbate 85 (TWEEN TM 85)TRITON TM N-101, TRITON TM X-100, oxtoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol H 15), polyoxyethylene-35-ricinoleate Soy lecithin and poloxamer. In a specific embodiment, the surfactant is polysorbate 80. In some of the embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.0001% to 10% polysorbate 80 by weight (w / w). In some of the embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.001% to 1% polysorbate 80 by weight (w / w). In some of the embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.01% to 1% polysorbate 80 by weight (w / w). In other embodiments, the final concentration of polysorbate 80 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1% polysorbate 80 (w / w). In another embodiment, the final concentration of polysorbate 80 in the formulation is 1% polysorbate 80 (w / w).

[0492] In a specific embodiment, the surfactant is polysorbate 20. In some of these embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.0001% to 10% polysorbate 20 by weight (w / w). In some of these embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.001% to 1% polysorbate 20 by weight (w / w). In some of these embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.01% to 1% polysorbate 20 by weight (w / w). In other embodiments, the final concentration of polysorbate 20 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% polysorbate 20 (w / w). In another embodiment, the final concentration of polysorbate 20 in the formulation is 1% polysorbate 20 (w / w).

[0493] In a specific embodiment, the surfactant is polysorbate 40. In some of these embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.0001% to 10% polysorbate 40 by weight (w / w). In some of these embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.001% to 1% polysorbate 40 by weight (w / w). In some of these embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.01% to 1% polysorbate 40 by weight (w / w). In other embodiments, the final concentration of polysorbate 40 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% polysorbate 40 (w / w). In another embodiment, the final concentration of polysorbate 40 in the formulation is 1% polysorbate 40 (w / w).

[0494] In a specific embodiment, the surfactant is polysorbate 60. In some of these embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.0001% to 10% polysorbate 60 by weight (w / w). In some of these embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.001% to 1% polysorbate 60 by weight (w / w). In some of these embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.01% to 1% polysorbate 60 by weight (w / w). In other embodiments, the final concentration of polysorbate 60 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% polysorbate 60 (w / w). In another embodiment, the final concentration of polysorbate 60 in the formulation is 1% polysorbate 60 (w / w).

[0495] In a specific embodiment, the surfactant is polysorbate 65. In some of these embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.0001% to 10% polysorbate 65 by weight (w / w). In some of these embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.001% to 1% polysorbate 65 by weight (w / w). In some of these embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.01% to 1% polysorbate 65 by weight (w / w). In other embodiments, the final concentration of polysorbate 65 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% polysorbate 65 (w / w). In another embodiment, the final concentration of polysorbate 65 in the formulation is 1% polysorbate 65 (w / w).

[0496] In a specific embodiment, the surfactant is polysorbate 85. In some of these embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.0001% to 10% polysorbate 85 by weight (w / w). In some of these embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.001% to 1% polysorbate 85 by weight (w / w). In some of these embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.01% to 1% polysorbate 85 by weight (w / w). In other embodiments, the final concentration of polysorbate 85 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% polysorbate 85 (w / w). In another embodiment, the final concentration of polysorbate 85 in the formulation is 1% polysorbate 85 (w / w).

[0497] In certain embodiments, the immunogenic compositions of the present disclosure have a pH of 5.5 to 7.5, more preferably a pH of 5.6 to 7.0, even more preferably a pH of 5.8 to 6.0.

[0498] In one embodiment, the present disclosure provides a container filled with any one of the immunogenic compositions disclosed herein. In one embodiment, the container is selected from the group consisting of a vial, a syringe, a flask, a fermenter, a bioreactor, a bag, a jar, an ampoule, a box, and a disposable pen. In certain embodiments, the container is siliconized.

[0499] In one embodiment, the container of the present disclosure is made of glass, metal (e.g., steel, stainless steel, aluminum, etc.) and / or polymer (e.g., thermoplastic, elastomer, thermoplastic elastomer). In one embodiment, the container of the present disclosure is made of glass.

[0500] In one embodiment, the present disclosure provides a syringe filled with any one of the immunogenic compositions disclosed herein. In certain embodiments, the syringe is siliconized and / or made of glass.

[0501] A typical dose of the immunogenic composition of the invention for injection has a volume of 0.1 mL to 2 mL, more preferably 0.2 mL to 1 mL, even more preferably a volume of about 0.5 mL.

[0502] 2.3 Use as antigen

[0503] The polysaccharides purified by the methods of the invention and the conjugates disclosed herein can be used as antigens. For example, they can be part of a vaccine.

[0504] Thus, in one embodiment, the polysaccharide purified by the method of the invention or the glycoconjugate obtained using the polysaccharide is used to generate an immune response in an individual. In one aspect, the individual is a mammal, such as a human, cat, sheep, pig, horse, cow or dog. In one aspect, the individual is a human.

[0505] In one embodiment, the polysaccharide purified by the method of the present invention, the glycoconjugate obtained using the polysaccharide, or the immunogenic composition disclosed herein is used in a vaccine.

[0506] In one embodiment, the polysaccharide purified by the method of the present invention, the glycoconjugate obtained using the polysaccharide, or the immunogenic composition disclosed herein is used as a medicament.

[0507] The immunogenic compositions described herein can be used in various therapeutic or prophylactic methods to prevent, treat or improve bacterial infections, diseases or conditions in individuals. Specifically, the immunogenic compositions described herein can be used to prevent, treat or improve Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Haemophilus influenzae type b, Escherichia coli, Neisseria meningitidis, Streptococcus agalactiae or Klebsiella pneumoniae infections, diseases or conditions in individuals.

[0508] Therefore, in one aspect, the present disclosure provides a method for preventing, treating or ameliorating an infection, disease or condition associated with Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Haemophilus influenzae type b, Escherichia coli, Neisseria meningitidis, Streptococcus agalactiae or Klebsiella pneumoniae in an individual, comprising administering to the individual an immunologically effective amount of an immunogenic composition of the present disclosure (particularly an immunogenic composition comprising a corresponding polysaccharide or a glycoconjugate thereof).

[0509] In one embodiment, the present disclosure provides a method of inducing an immune response in a subject to Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Haemophilus influenzae type b, Escherichia coli, Neisseria meningitidis, Streptococcus agalactiae or Klebsiella pneumoniae, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present invention (particularly an immunogenic composition comprising a corresponding polysaccharide or a glycoconjugate thereof).

[0510] In one embodiment, the immunogenic compositions disclosed herein are used as vaccines. In such embodiments, the immunogenic compositions described herein can be used to prevent Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Haemophilus influenzae type b, Escherichia coli, Neisseria meningitidis, or Streptococcus agalactiae infection in an individual. Therefore, in one aspect, the present disclosure provides a method of preventing infection by Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Haemophilus influenzae type b, Escherichia coli, Neisseria meningitidis, Streptococcus agalactiae, or Klebsiella pneumoniae in an individual, comprising administering to the individual an immunologically effective amount of the immunogenic composition of the present invention.

[0511] In one aspect, the subject is a mammal, such as a human, cat, sheep, pig, horse, cow or dog. In one aspect, the subject is a human.

[0512] The immunogenic compositions disclosed herein can be used to protect or treat humans susceptible to infection with Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Haemophilus influenzae type b, Escherichia coli, Neisseria meningitidis, Streptococcus agalactiae, or Klebsiella pneumoniae by administering the immunogenic compositions by systemic or mucosal routes. In one embodiment, the immunogenic compositions disclosed herein are administered by intramuscular, intraperitoneal, intradermal, or subcutaneous routes. In one embodiment, the immunogenic compositions disclosed herein are administered by intramuscular, intraperitoneal, intradermal, or subcutaneous injection. In one embodiment, the immunogenic compositions disclosed herein are administered by intramuscular or subcutaneous injection.

[0513] In some cases, only as little as one dose of an immunogenic composition according to the present disclosure is required, but in some cases, such as cases of greater immunodeficiency, a second, third or fourth dose may be given. After the initial vaccination, the individual may receive one or more adequately spaced booster immunizations.

[0514] In one embodiment, the vaccination schedule of the immunogenic composition according to the present disclosure is a single dose.

[0515] In one embodiment, the vaccination schedule of the immunogenic composition according to the present disclosure is a multiple-dose schedule.

[0516] 3. Sugars from E. coli

[0517] In one embodiment, the carbohydrate is produced in recombinant Gram-negative bacteria. In one embodiment, the carbohydrate is produced in recombinant E. coli cells. In one embodiment, the carbohydrate is produced in recombinant Salmonella cells. Exemplary bacteria include E. coli O25K5H1, E. coli BD559, E. coli GAR2831, E. coli GAR865, E. coli GAR868, E. coli GAR869, E. coli GAR872, E. coli GAR878, E. coli GAR896, E. coli GAR1902, E. coli O25a ETC NR-5, E. coli O157:H7:K-, Salmonella enterica serotype typhimurium strain LT2, E. coli GAR2401, Salmonella enterica serotype Enteritidis CVD 1943, Salmonella enterica serotype Typhimurium CVD 1925, Salmonella enterica serotype Paratyphi A CVD 1902, and Shigella flexneri CVD 1208S. In one embodiment, the bacterium is not E. coli GAR2401. This genetic approach to carbohydrate production allows for the efficient production of O-polysaccharide and O-antigen molecules as vaccine components.

[0518] As used herein, the term "wzz protein" refers to a polypeptide having a chain length, such as wzzB, wzz, wzz SF 、wzz ST 、fepE、wzz fepE, wzz1 and wzz2. The GenBank accession numbers of exemplary wzz gene sequences are AF011910 for E4991 / 76, AF011911 for F186, AF011912 for M70 / 1-1, AF011913 for 79 / 311, AF011914 for Bi7509-41, AF011915 for C664-1992, AF011916 for C258-94, AF011917 for C722-89, and AF011919 for EDL933. The GenBank accession numbers of G7 and Bi316-41 wzz gene sequences are U39305 and U39306, respectively. Other GenBank accession numbers for exemplary wzz gene sequences are NP_459581 for Salmonella enterica subspecies serovar Typhimurium strain LT2 FepE; AIG66859 for E. coli O157:H7 strain EDL933 FepE; NP_461024 for Salmonella enterica subspecies serovar Typhimurium strain LT2 WzzB, NP_416531 for E. coli K-12 substrain MG1655 WzzB, and NP_415119 for E. coli K-12 substrain MG1655 FepE. In a preferred embodiment, the wzz family proteins are wzzB, wzz, wzz SF 、wzz ST 、fepE、wzz fepE , wzz1 and wzz2, most preferably wzzB, more preferably fepE.

[0519] Exemplary wzzB sequences include:

[0520] >O25b 2401 WzzB

[0521] MRVENNNVSGQNHDPEQIDLIDLLVQLWRGKMTIIISVIVAIALAIGYLAVAKEKWTSTAIITQPDVGQIAGYNNAMNVIYGQAAPKVSDLQETLIGRFSSAFSALAETLDNQEEPEKLTIEPSVKNQQLPLTVSYVGQTAEGAQMKLAQYIQQVDDKVNQELE KDLKDNIALGRKNLQDSLRTQEVVAQEQKDLRIRQIQEALQYANQEQVTKPQVQQTEDVTQDTLFLLGSEALESMIKHEATRPLVFSSNYYQTRQNLLDIESLKVDDLDIHAYRYVMKPTLPIRRDSPKKAITLILAVLLGGMVGAGIVLGRNALRNYNAK(SEQ ID NO:20)

[0522] >O25a:K5:H1 WzzB

[0523] MRVENNNVSGQNDPEQIDLIDLLVQLWRGKMTIIISVIVAIALAIGYLAVAKEKWTSTAIITQPDVGQIAGYNNAMNVIYGQAAPKVSDLQETLIGRFSASAFSALAETLDNQDEPEKLTIEPSVKNQLPLTVSYVGQTAEGAQMKQQQQQVKVKVKVK DLKDNIALGRKNLQDSLRTQEVVAQEQKDLRIRQIQIQYANQAQVTKPQIQTGEDITQDTLFLLGSEALESMIKHEATRPLVFSPNYYQTRQNLLDIESLKVDDLDIHAYRYVMKPTLPIRRDSPKAITLILAVGLLGGLGVLGAVLQLGNAQLQLQLGDITQDTLFLLGSEALESMIKHEATRPLVFSPNYYQTRQNLLDIESL ID NO:21)

[0524] >O25a ETEC ATCC WzzB

[0525] MRVENNNVSGQNHDPEQIDLIDLLVQLWRGKMTIIISVVVAIALAIGYLAVAKEKWTSTAIITQPDVGQIAGYNNAMNVIYGQAAPKVSDLQETLIGRFSFAFSALAETLDNQKEPEKLTIEPSVKNQLPLTVSYVGQTAEDAQMKQQQQQQQQLVKVGQIAGYNNAMNVIYGQAAPKVSDLQETLIGRFSFAFSALAETLDNQKEPEKLTIEPSVKNQLPLTVSYVGQTAEDAQMQQQQQQQQQLVGQIAGYNNAMN DLKDNLALGRKNLQDSLRTQEVVAQEQKDLRIRQIQEALQYANQAQVTKPQIQTGEDITQDTLFLLGSEALESMIKHEATRPLVFSPNYYQTRQNLLDIENLKVDDLDIHAYRYVMKPTLPIRRDSPKKAITLILAVLGGLGLGVLQLQLQLQLQLGEDITQDTLFLLGSEALESMIKHEATRPLVFSPNYYQTRQNLLDIENLKVDDDLDIHAYRYVMKPTLPIRRDSPKAITLILAVLGLGGLGVLLQLQLQLQLQL ID NO:22)

[0526] >K12 W3110 WzzB

[0527] MRVENNNVSGQNHDPEQIDLIDLLVQLWRGKMTIIISVIVAIALAIGYLAVAKEKWTSTAIITQPDVGQIAGYNNAMNVIYGQAAPKVSDLQETLIGRFSSAFSALAETLDNQEEREKLTIEPSVKNQQLPLTVSYVGQTAEGAQMKLAQYIQQVDDKVNQELEKDLKDNIALGRKNLQDSLRTQEVVAQEQKDLRIRQIQEALQYANQAQVTKPQIQQTGEDITQDTLFLLGSEALESMIKHEATRPLVFSPNYYQTRQNLLDIESLKVDDLDIHAYRYVMKPMLPIRRDSPKKAITLILAVLLGGMVGAGIVLGRNALRNYNAK(SEQ ID NO:23)

[0528] >Salmonella LT2 WzzB

[0529] MTVDSNTSSGRGNDPEQIDLIELLLQLWRGKMTIIVAVIIAILLAVGYLMIAKEKWTSTAIITQPDAAQVATYTNALNVLYGGNAPKISEVQANFISRFSSAFSALSEVLDNQKEREKLTIEQSVKGQALPLSVSYVSTTAEGAQRRLAEYIQQVDEEVAKELEVDLKDNITLQTKTLQESLETQEVVAQEQKDLRIKQIEEALRYADEAKITQPQIQQTQDVTQDTMFLLGSDALKSMIQNEATRPLVFSPAYYQTKQTLLDIKNLKVTADTVHVYRYVMKPTLPVRRDSPKTAITLVLAVLLGGMIGAGIVLGRNALRSYKPKAL(SEQ ID NO:24)

[0530] Exemplary FepE sequences include:

[0531] >O25b GAR2401 FepE

[0532] MSSLNIKQGSDAHFPDYPLASPSSNNEIDLLNLISVLWRAKKTVMAVVFAFACAGLLISFILPQKWTCAAVVTPPEPVQWQELEKSFTKLRVLDLD IKIDRTEAFNLFIKFQSVSLLEYLRSSPYVMDQLKEAKIDELHRAIVALSEKMKAVDDNASKKKDEPSLYTSWTLSFTAPTSEEAQTVLSG YIDYISTLVVKESLENVRNKLEIKTQFEKEKLAQDRIKTKNQLDANIQRLNYSLDIANAGIKKPVYSNGQDDPDFSISLGADGIERKLEIE KAVTDVAELNGELRNRQYLVEQLTKAHVNDVNFTPFKYQLSPSLPVKKDGPGKAIIVILSALIGGMVACGGVLLRYAMASRKQDAMMADHLV(SEQ ID NO:15)

[0533] >O25a:K5:H1 Fever

[0534] MSSLNIKQGSEAHFPEYPLASPSNNEIDLLNLIEVLWRAKKTVMAVVFAFACAGLLISFILPQKWTCAAVVTPPEPVQWQELEKTFTKLRVLDLD IKIDRTEAFNLFIKFQSVSLLEEYLRSSPYVMDQLKEAKIDPLDLHRAIVALSEKMKAVDDNASKKKDESALYTSWTLSFTAPTSEEAQKVLAG YIDYISALVVKESIENVRNKLEIKTQFEKEKLAQDRIKTKNQLDANIQRLNYSLDIANAAGIKKPVYSNGQADDPDFSISLGADGIERKLEIE KAVTDVAELNGELNRQYLVEQLTKTNINDVNFTPFKYQLRPSLPVKKDGQGKAIIVILSALVGGMVACGGVLLRHAMASRKQDAMMADHLV(SEQ ID NO:16)

[0535] >O25a ETEC ATCC FepE

[0536] MSSLNIKQGSDAHFPDYPLASPSNNEIDLLNLISVLWRAKKTVMAVVFAFACAGLLISFILPQKWTSAAVVTPPEPVQWQELEKSFTKLRVLDLDIKIDRTEAFNLFIKKFQSVSLLEEYLRSSPYVMDQLKEAKIDELDLHRAIVALSEKMKAVDDNASKKKDEPSLYTSWTLSFTAPTSEEAQTVLSGYIDYISTLVVKESLENVRNKLEIKTQFEKEKLAQDRIKTKNQLDANIQRLNYSLDIANAAGIKKPVYSNGQAVKDDPDFSISLGADGIERKLEIEKAVTDVAELNGELRNRQYLVEQLTKAHVNDVNFTPFKYQLSPSLPVKKDGPGKAIIVILSALIGGMVACGGVLLRYAMASRKQDAMMADHLV(SEQ ID NO:17)

[0537] >O157 FepE

[0538] MSSLNIKQGSDAHFPDYPLASPSNNEIDLLNLISVLWRAKKTVMAVVFAFACAGLLISFILPQKWTSAAVVTPPEPVQWQELEKTFTKLRVLDLDIKIDRTEAFNLFIKKFQSVSLLEEYLRSSPYVMDQLKEAKIDELDLHRAIVALSEKMKAVDDNASKKKDEPSLYTSWTLSFTAPTSEEAQTVLSGYIDYISALVVKESIENVRNKLEIKTQFEKEKLAQDRIKMKNQLDANIQRLNYSLDIANAAGIKKPVYSNGQAVKDDPDFSISLGADGIERKLEIEKAVTDVAELNGELRNRQYLVEQLTKANINDVNFTPFKYQLSPSLPVKKDGPGKAIIVILSALIGGMVACGSVLLRYAMASRKQDAMMADHLV(SEQ ID NO:18)

[0539] >Salmonella LT2 FepE

[0540] MPSLNVKQEKNQSFAGYSLPPANSHEIDLFSLIEVLWQAKRRILATVFAFACVGLLLSFLLPQKWTSQAIVTPAESVQWQGLERTLTALRVLDMEVSVDRGSVFNLFIKKFSSPSLLEEYLRSSPYVMDQLKGAQIDEQDLHRAIVLLSEKMKAVDSNVGKKNETSLFTSWTLSFTAPTREEAQKVLAGYI QYISDIVVKETLENIRNQLEIKTRYEQEKLAMDRVRLKNQLDANIQRLHYSLEIANAAGIKRPVYSNGQAVKDDPDFSISLGADGISRKLEIEKGVTDVAEIDGDLRNRQYHVEQLAAMNVSDVKFTPFKYQLSPSLPVKKDGPGKAIIIILAALIGGMMACGGVLLRHAMVSRKMENALAIDERLV(SEQ ID NO:19)

[0541] In some embodiments, modified carbohydrates (modified compared to the corresponding wild-type carbohydrates) can be produced by expressing (not necessarily overexpressing) a wzz family protein (e.g., fepE) from a gram-negative bacterium in a gram-negative bacterium and / or by turning off (i.e., inhibiting, deleting, removing) a second wzz gene (e.g., wzzB) to produce high molecular weight carbohydrates containing medium or long O-antigen chains, such as lipopolysaccharides. For example, the modified carbohydrates can be produced by expressing (not necessarily overexpressing) wzz2 and turning off wzz1. Alternatively, in an alternative, the modified carbohydrates can be produced by expressing (not necessarily overexpressing) wzzfepE and turning off wzzB. In another embodiment, the modified carbohydrates can be produced by expressing (not necessarily overexpressing) wzzB but turning off wzzfepE. In another embodiment, the modified carbohydrates can be produced by expressing fepE. Preferably, the wzz family proteins are derived from strains that are heterologous to the host cell.

[0542] In one aspect, the present invention relates to carbohydrates produced by expressing wzz family proteins, preferably fepE, in Gram-negative bacteria to produce high molecular weight carbohydrates containing medium or long O-antigen chains, which have an increase of at least 1, 2, 3, 4 or 5 repeating units compared to the corresponding wild-type O-polysaccharide. In one aspect, the present invention relates to carbohydrates produced by Gram-negative bacteria in cultures expressing (not necessarily overexpressing) wzz family proteins (e.g., wzzB) from Gram-negative bacteria to produce high molecular weight carbohydrates containing medium or long O-antigen chains, which have an increase of at least 1, 2, 3, 4 or 5 repeating units compared to the corresponding wild-type O-antigen. For other exemplary carbohydrates with an increased number of repeating units compared to the corresponding wild-type carbohydrate, see the description of O-polysaccharides and O-antigens below. The desired chain length is the chain length that produces improved or maximum immunogenicity in a given vaccine construct.

[0543] In another embodiment, the saccharide comprises any one of the formulas selected from Table 1, wherein the number of repeating units n in the saccharide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 200, 201, 202, 203, 204, 205 Preferably, the saccharide comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 repeating units compared to the corresponding wild-type O-polysaccharide. Methods for determining the length of saccharides are known in the art. Such methods include nuclear magnetic resonance, mass spectrometry and size exclusion chromatography.

[0544] In a preferred embodiment, the invention relates to carbohydrates produced in recombinant E. coli host cells, wherein the gene for the endogenous wzz O-antigen length regulator (e.g., wzzB) is deleted and replaced by a (second) wzz gene from a Gram-negative bacterium (e.g., Salmonella fepE) that is heterologous to the recombinant E. coli host cell, to produce high molecular weight carbohydrates, such as lipopolysaccharides, containing medium or long O-antigen chains. In some embodiments, the recombinant E. coli host cell comprises a wzz gene from Salmonella, preferably from Salmonella enterica.

[0545] In one embodiment, the host cell comprises a heterologous gene of a wzz family protein as a stably maintained plasmid vector. In another embodiment, the host cell comprises a heterologous gene of a wzz family protein as an integrated gene in the host cell chromosomal DNA. Methods for stably expressing plasmid vectors in E. coli host cells and methods for integrating heterologous genes into the chromosome of an E. coli host cell are known in the art. In one embodiment, the host cell comprises a heterologous gene of an O-antigen as a stably maintained plasmid vector. In another embodiment, the host cell comprises a heterologous gene of an O-antigen as an integrated gene in the host cell chromosomal DNA. Methods for stably expressing plasmid vectors in E. coli host cells and Salmonella host cells are known in the art. Methods for integrating heterologous genes into the chromosomes of E. coli host cells and Salmonella host cells are known in the art.

[0546] In one aspect, the recombinant host cell is cultivated in a culture medium comprising a carbon source. The carbon source for cultivating Escherichia coli is known in the art. Exemplary carbon sources include sugar alcohols, polyols, alditols or ketoses, including but not limited to arabinose, cellobiose, fructose, glucose, glycerine, inositol, lactose, maltose, mannitol, mannose, rhamnose, raffinose, sorbitol, sorbose, sucrose, trehalose, pyruvic acid, succinate and methylamine. In a preferred embodiment, the culture medium includes glucose. In certain embodiments, the culture medium includes polyols or alditols, such as mannitol, inositol, sorbose, glycerine, sorbitol, lactose and arabinose as carbon sources. All carbon sources can be added to the culture medium before the start of cultivation, or can be progressively added or continuously added during the incubation period.

[0547] An exemplary culture medium for recombinant host cells comprises an ingredient selected from any of the following: KH 2 PO 4 , K 2 HPO 4 NH 4 ) 2 SO 4 , sodium citrate, Na 2 SO 4, aspartic acid, glucose, MgSO 4 、FeSO 4 -7H 2 O、Na 2 MoO 4 -2H 2 O, H 3 BO 3 、CoCl 2 -6H 2 O, CuCl 2 -2H 2 O、MnCl 2 -4H 2 O、ZnCl 2 and CaCl 2 -2H 2 O. Preferably, the culture medium contains KH 2 PO 4 , K 2 HPO 4 NH 4 ) 2 SO 4 , sodium citrate, Na 2 SO 4 , aspartic acid, glucose, MgSO 4 、FeSO 4 -7H 2 O、Na 2 MoO 4 -2H 2 O, H 3 BO 3 、CoCl 2 -6H 2 O, CuCl 2 -2H 2 O、MnCl 2 -4H 2 O、ZnCl 2 and CaCl 2 -2H 2 O.

[0548] The culture medium used herein may be solid or liquid, synthetic (ie, artificial) or natural, and may contain sufficient nutrients for cultivating recombinant host cells. Preferably, the culture medium is a liquid culture medium.

[0549] In certain embodiments, the culture medium may also include suitable inorganic salts. In certain embodiments, the culture medium may also include micronutrients. In certain embodiments, the culture medium may also include growth factors. In certain embodiments, the culture medium may also include additional carbon sources. In certain embodiments, the culture medium may also include suitable inorganic salts, micronutrients, growth factors and supplementary carbon sources. Inorganic salts, micronutrients, growth factors and supplementary carbon sources suitable for cultivating Escherichia coli are known in the art.

[0550] In some embodiments, the culture medium may optionally contain additional components such as peptone, NZ amine, enzymatic soy hydride, additional yeast extract, malt extract, supplementary carbon source, and various vitamins. In some embodiments, the culture medium does not contain such additional components such as peptone, NZ amine, enzymatic soy hydride, additional yeast extract, malt extract, supplementary carbon source, and various vitamins.

[0551] Illustrative examples of suitable supplemental carbon sources include, but are not limited to, other carbohydrates such as glucose, fructose, mannitol, starch or starch hydrolysates, cellulose hydrolysates, and molasses; organic acids such as acetic acid, propionic acid, lactic acid, formic acid, malic acid, citric acid, and fumaric acid; and alcohols such as glycerol, inositol, mannitol, and sorbitol.

[0552] In some embodiments, the culture medium further comprises a nitrogen source. Nitrogen sources suitable for culturing E. coli are known in the art. Illustrative examples of suitable nitrogen sources include, but are not limited to, ammonia, including ammonia gas and ammonia water; ammonium salts of inorganic or organic acids, such as ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate, and ammonium acetate; urea; nitrates or nitrites and other nitrogen-containing substances, including pure or crude amino acids, meat extracts, peptones, fish meal, fish hydrolysates, corn steep liquor, casein hydrolysates, soybean cake hydrolysates, yeast extracts, dry yeast, ethanol yeast distillates, soybean meal, cottonseed meal, etc.

[0553] In some embodiments, the culture medium comprises inorganic salts. Illustrative examples of suitable inorganic salts include, but are not limited to, potassium salts, calcium salts, sodium salts, magnesium salts, manganese salts, iron salts, cobalt salts, zinc salts, copper salts, molybdenum salts, tungsten salts and other trace element salts, and phosphates.

[0554] In some embodiments, the culture medium comprises appropriate growth factors. Illustrative examples of appropriate micronutrients, growth factors, etc. include, but are not limited to, coenzyme A, pantothenic acid, pyridoxine-HCl, biotin, thiamine, riboflavin, flavin mononucleotide, flavin adenine dinucleotide, DL-6,8-lipoic acid, folic acid, vitamin B12, vitamin B13, vitamin B14, vitamin B15, vitamin B16, vitamin B17, vitamin B18, vitamin B19, vitamin B20, vitamin B21, vitamin B22, vitamin B23, vitamin B24, vitamin B25, vitamin B26, vitamin B27, vitamin B28, vitamin B29, vitamin B2A, vitamin B2B, vitamin B2C, vitamin B2C, vitamin B2A, vitamin B2B ... 12, other vitamins, amino acids such as cysteine ​​and hydroxyproline, bases such as adenine, uracil, guanine, thymine and cytosine, sodium thiosulfate, p-aminobenzoic acid or r-aminobenzoic acid, nicotinamide, nitriloacetic acid esters, etc., as pure or partially purified compounds or present in natural materials. The amount can be determined empirically by those skilled in the art according to methods and techniques known in the art.

[0555] In another embodiment, the modified carbohydrates described herein (compared to the corresponding wild-type carbohydrates) are synthetically produced, for example, in vitro. The synthetic production or synthesis of carbohydrates can help avoid cost and time-intensive production processes. In one embodiment, carbohydrates are synthesized synthetically, for example, by using a sequential glycosylation strategy or a combination of sequential glycosylation and [3+2] block synthesis strategies, synthesized from appropriately protected monosaccharide intermediates. For example, thioglycosides and glycosyl trichloroacetimidate derivatives can be used as glycosyl donors in glycosylation. In one embodiment, carbohydrates synthesized synthetically in vitro have the same structure as carbohydrates produced by recombinant means, such as by operating the above-mentioned wzz family proteins.

[0556] Saccharides produced (recombinantly or synthetically) include structures derived from any E. coli serotype, including, for example, any of the following E. coli serotypes: O1 (e.g., O1A, O1B, and O1C), O2, O3, O4 (e.g., O4:K52 and O4:K6), O5 (e.g., O5ab and O5ac (strain 180 / C3)), O6 (e.g., O6:K2; K13; K15 and O6:K54), O7, O8, O9, O10, O11, O12, O13, O14, O15, O16, O17, O18 (e.g., O18A, O18ac, O18A1), , O18B and O18B1), O19, O20, O21, O22, O23 (e.g., O23A), O24, O25 (e.g., O25a and O25b), O26, O27, O28, O29, O30, O32, O33, O34, O35, O36, O37, O38, O39, O40, O41, O42, O43, O44, O45 (e.g., O45 and O45rel), O46, O48, O49, O50, O51, O52, O53, O54, O55, O56, O57, O58, O59, O60, O61, O62, O62D 1, O63, O64, O65, O66, O68, O69, O70, O71, O73 (e.g., O73 (strain 73-1)), O74, O75, O76, O77, O78, O79, O80, O81, O82, O83, O84, O85, O86, O87, O88, O89, O90, O91, O92, O93, O95, O96, O97, O98 8. O99, O100, O101, O102, O103, O104, O105, O106, O107, O108, O109, O110, 0111, O112, O1 13. O114, O115, O116, O117, O118, O119, O120, O121, O123, O124, O125, O126, O127, O128, O 129, O130, O131, O132, O133, O134, O135, O136, O137, O138, O139, O140, O141, O142, O143 , O144, O145, O146, O147, O148, O149, O150, O151, O152, O153, O154, O155, O156, O157, O15 8. O159, O160, O161, O162, O163, O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186 and O187.

[0557] Individual polysaccharides are usually purified (enriched in terms of the amount of polysaccharide-protein conjugate) by methods known in the art, such as dialysis, concentration procedures, diafiltration procedures, tangential flow filtration, precipitation, elution, centrifugation, precipitation, ultrafiltration, depth filtration and / or column chromatography (ion exchange chromatography, multimodal ion exchange chromatography, DEAE and hydrophobic interaction chromatography). Preferably, the polysaccharide is purified by a method comprising tangential flow filtration.

[0558] The purified polysaccharide can be activated (eg, chemically activated) to render it reactive (eg, directly with a carrier protein or via a linking group such as an eTEC spacer), and subsequently incorporated into a glycoconjugate of the invention, as further described herein.

[0559] In a preferred embodiment, the saccharide of the present invention is derived from an E. coli serotype, wherein the serotype is O25a. In another preferred embodiment, the serotype is O25b. In another preferred embodiment, the serotype is O1A. In another preferred embodiment, the serotype is O2. In another preferred embodiment, the serotype is O6. In another preferred embodiment, the serotype is O17. In another preferred embodiment, the serotype is O15. In another preferred embodiment, the serotype is O18A. In another preferred embodiment, the serotype is O75. In another preferred embodiment, the serotype is O4. In another preferred embodiment, the serotype is O16. In another preferred embodiment, the serotype is O13. In another preferred embodiment, the serotype is O7. In another preferred embodiment, the serotype is O8. In another preferred embodiment, the serotype is O9.

[0560] As used herein, reference to any of the serotypes listed above refers to serotypes that encompass repeating unit structures (O-units, described below) known in the art and that are unique to the corresponding serotype. For example, the term "O25a" serotype (also referred to in the art as serotype "O25") refers to serotypes that encompass the formula O25 shown in Table 1. As another example, the term "O25b" serotype refers to serotypes that encompass the formula O25b shown in Table 1.

[0561] As used herein, unless otherwise indicated, serotypes are referred to collectively herein, for example, the term formula "O18" is generally meant to encompass formula O18A, formula O18ac, formula 18A1, formula O18B, and formula O18B1.

[0562] As used herein, the term "O1" generally refers to the species encompassing the generic term "O1" in the formula name according to Table 1, such as any of Formula O1A, Formula O1A1, Formula O1B, and Formula O1C, each of which is shown in Table 1. Thus, "O1 serotype" generally refers to the serotype encompassing any of Formula O1A, Formula O1A1, Formula O1B, and Formula O1C.

[0563] As used herein, the term "O6" generally refers to the species of formulas that include the generic term "O6" in the formula names according to Table 1, such as any of the formulas O6:K2, K13, K15, and O6:K54, each of which is shown in Table 1. Thus, "O6 serotype" generally refers to a serotype encompassing any of the formulas 6:K2, K13, K15, and O6:K54.

[0564] Other examples of terms generally referring to the class of formulae containing the generic term according to the formula names of Table 1 include: "O4", "O5", "O18" and "O45".

[0565] As used herein, the term "O2" refers to the formula O2 shown in Table 1. The term "O2 O-antigen" refers to a saccharide encompassing the formula O2 shown in Table 1.

[0566] As used herein, reference to O-antigens from the serotypes listed above is meant to encompass saccharides of the formula labeled with the corresponding serotype name. For example, the term "O25B O-antigen" is meant to encompass saccharides of formula O25B shown in Table 1.

[0567] As another example, the term "O1 O-antigen" is generally meant to encompass carbohydrates of formulas comprising the term "O1", such as Formula O1A, Formula O1A1, Formula O1B, and Formula O1C, each of which is shown in Table 1.

[0568] As another example, the term "O6 O-antigen" is generally meant to encompass carbohydrates of formulas including the term "O6", such as Formula O6:K2, Formula O6:K13, Formula O6:K15, and Formula O6:K54, each of which is shown in Table 1.

[0569] O-polysaccharides

[0570] As used herein, the term "O-polysaccharide" refers to any structure comprising an O-antigen, so long as the structure does not comprise whole cells or lipid A. For example, in one embodiment, the O-polysaccharide comprises lipopolysaccharide, in which lipid A is not bound. Steps for removing lipid A are known in the art and include, for example, heat treatment with the addition of an acid. Exemplary methods include treatment with 1% acetic acid at 100°C for 90 minutes. This method is combined with a method to separate lipid A as it is removed. Exemplary methods for separating lipid A include ultracentrifugation.

[0571] In one embodiment, O-polysaccharide refers to a structure consisting of O-antigen, in which case O-polysaccharide is synonymous with the term O-antigen. In a preferred embodiment, O-polysaccharide refers to a structure comprising repeating units of O-antigen but without a core sugar. Thus, in one embodiment, O-polysaccharide does not comprise the E. coli R1 core portion. In another embodiment, O-polysaccharide does not comprise the E. coli R2 core portion. In another embodiment, O-polysaccharide does not comprise the E. coli R3 core portion. In another embodiment, O-polysaccharide does not comprise the E. coli R4 core portion. In another embodiment, O-polysaccharide does not comprise the E. coli K12 core portion. In another preferred embodiment, O-polysaccharide refers to a structure comprising O-antigen and a core sugar. In another embodiment, O-polysaccharide refers to a structure comprising O-antigen, a core sugar and a KDO portion.

[0572] Methods for purifying O-polysaccharides including core oligosaccharides from LPS are known in the art. For example, after LPS purification, the purified LPS can be hydrolyzed by heating in 1% (v / v) acetic acid at 100 degrees Celsius for 90 minutes, followed by ultracentrifugation at 142,000×g for 5 hours at 4 degrees Celsius. The supernatant containing the O-polysaccharide is freeze-dried and stored at 4 degrees Celsius. In certain embodiments, deletion of capsule synthesis genes that enable simple purification of O-polysaccharides is described.

[0573] O-polysaccharide can be isolated by methods including, but not limited to, mild acid hydrolysis to remove lipid A from LPS. Other embodiments may include using hydrazine as a reagent for preparing O-polysaccharide. Preparation of LPS can be accomplished by methods known in the art.

[0574] In certain embodiments, O-polysaccharides purified from wild-type, modified or attenuated Gram-negative bacterial strains expressing (not necessarily overexpressing) Wzz proteins (e.g., wzzB) are provided for use in conjugate vaccines. In a preferred embodiment, O-polysaccharide chains are purified from Gram-negative bacterial strains expressing (not necessarily overexpressing) wzz proteins for use as vaccine antigens, as conjugates or composite vaccines.

[0575] In one embodiment, the molecular weight of the O-polysaccharide is increased by about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold compared to the corresponding wild-type O-polysaccharide. In a preferred embodiment, the molecular weight of the O-polysaccharide is increased by at least 1-fold and at most 5-fold compared to the corresponding wild-type O-polysaccharide. In another embodiment, the molecular weight of the O-polysaccharide is increased by at least 2 times and at most 4 times compared to the corresponding wild-type O-polysaccharide. The increase in the molecular weight of the O-polysaccharide is preferably associated with an increase in the number of O-antigen repeating units compared to the corresponding wild-type O-polysaccharide. In one embodiment, the increase in the molecular weight of the O-polysaccharide is due to wzz family proteins.

[0576] In one embodiment, the molecular weight of the O-polysaccharide is increased by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100 kDa or more. In one embodiment, the molecular weight of the O-polysaccharide of the present invention is increased by at least 1 and at most 200 kDa compared to the corresponding wild-type O-polysaccharide. In one embodiment, the molecular weight is increased by at least 5 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 10 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 12 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 15 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 18 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 20 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 21 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 22 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 30 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 1 and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 5 and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 10 and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 12 and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 15 and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 20 and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 1 and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 5 and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10 and at most 75 kDa. In one embodiment, the molecular weight increases by at least 12 and at most 75 kDa. In one embodiment, the molecular weight increases by at least 15 and at most 75 kDa. In one embodiment, the molecular weight increases by at least 18 and at most 75 kDa. In one embodiment, the molecular weight increases by at least 20 and at most 75 kDa.In one embodiment, the molecular weight increases by at least 30 and at most 75 kDa. In one embodiment, the molecular weight increases by at least 10 and at most 90 kDa. In one embodiment, the molecular weight increases by at least 12 and at most 85 kDa. In one embodiment, the molecular weight increases by at least 10 and at most 75 kDa. In one embodiment, the molecular weight increases by at least 10 and at most 70 kDa. In one embodiment, the molecular weight increases by at least 10 and at most 60 kDa. In one embodiment, the molecular weight increases by at least 10 and at most 50 kDa. In one embodiment, the molecular weight increases by at least 10 and at most 49 kDa. In one embodiment, the molecular weight increases by at least 10 and at most 48 kDa. In one embodiment, the molecular weight increases by at least 10 and at most 47 kDa. In one embodiment, the molecular weight increases by at least 10 and at most 46 kDa. In one embodiment, the molecular weight increases by at least 20 and at most 45 kDa. In one embodiment, the molecular weight increases by at least 20 and at most 44 kDa. In one embodiment, the molecular weight increases by at least 20 and at most 43 kDa. In one embodiment, the molecular weight increases by at least 20 and at most 42 kDa. In one embodiment, the molecular weight increases by at least 20 and at most 41 kDa. Said increase in molecular weight of the O-polysaccharide is preferably associated with an increase in the number of O-antigen repeating units compared to the corresponding wild-type O-polysaccharide. In one embodiment, the increase in molecular weight of the O-polysaccharide is due to wzz family proteins.

[0577] In another embodiment, the O-polysaccharide comprises any one of the formulas selected from Table 1, wherein the number of repeating units n in the O-polysaccharide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 greater than the number of repeating units in the corresponding wild-type O-polysaccharide. , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more repeating units. Preferably, the saccharide comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 additional repeat units compared to the corresponding wild type O-polysaccharide.

[0578] O-antigen

[0579] O antigen is part of the lipopolysaccharide (LPS) of the outer membrane of Gram-negative bacteria. O antigen is located on the cell surface and is a variable cell component. The variability of O antigen provides the basis for serotyping of Gram-negative bacteria. The current E. coli serotyping scheme contains O-polysaccharides 1 to 181.

[0580] The O-antigen comprises an oligosaccharide repeating unit (O-unit), the wild-type structure of which typically contains two to eight residues from a wide range of carbohydrates. The O-units of exemplary E. coli O-antigens are shown in Table 1. The O-units of exemplary K. pneumoniae O-antigens are shown in Table 1a.

[0581] In one embodiment, the carbohydrate of the present invention can be an oligosaccharide unit. In one embodiment, the carbohydrate of the present invention is a repeating oligosaccharide unit of the relevant serotype. In such embodiments, the carbohydrate can comprise a structure selected from any one of formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O52, formula O97 and formula O101.

[0582] In one embodiment, the saccharide of the present invention can be an oligosaccharide. Oligosaccharides have a low number of repeating units (usually 5-15 repeating units) and are usually derived synthetically or by hydrolysis of polysaccharides. In such embodiments, the saccharide can include a structure selected from any one of formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O52, formula O97 and formula O101.

[0583] Preferably, all saccharides of the invention and all saccharides in the immunogenic compositions of the invention are polysaccharides. High molecular weight polysaccharides can induce certain antibody immune responses due to the epitopes present on the surface of the antigen. Preferably, the separation and purification of high molecular weight polysaccharides is contemplated for use in the conjugates, compositions and methods of the invention.

[0584] In some embodiments, the number of repeating O units in each individual O-antigen polymer (and therefore the length and molecular weight of the polymer chain) depends on the wzz chain length regulator, an inner membrane protein. Different wzz proteins confer different ranges of modal lengths (4 to> 100 repeat units). The term "modal length" refers to the number of repeating O units. Gram-negative bacteria typically have two different Wzz proteins that confer two different OAg modal chain lengths, one longer and one shorter. The expression (not necessarily overexpression) of wzz family proteins (e.g., wzzB) in Gram-negative bacteria can allow manipulation of O antigen length, change or bias bacteria to produce O antigens of certain length ranges, and increase the generation of high-yield high-molecular-weight lipopolysaccharides. In one embodiment, "short" modal length as used herein refers to a low number of repeating O- units, such as 1-20. In one embodiment, "long" modal length as used herein refers to a number of repeating O- units greater than 20 and at most 40. In one embodiment, "very long" modal length as used herein refers to greater than 40 repeating O- units.

[0585] In one embodiment, the generated saccharide has at least 10 repeat units, 15 repeat units, 20 repeat units, 25 repeat units, 30 repeat units, 35 repeat units, 40 repeat units, 45 repeat units, 50 repeat units, 55 repeat units, 60 repeat units, 65 repeat units, 70 repeat units, 75 repeat units, 80 repeat units, 85 repeat units, 90 repeat units, 95 repeat units or 100 repeat units increased compared to the corresponding wild-type O-polysaccharide.

[0586] In another embodiment, the saccharide of the invention has an increase of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 94, 95, 96, 97, 98, 99, 100 or more repeating units. Preferably, the saccharide comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 additional repeat units compared to the corresponding wild type O-polysaccharide.

[0587] Methods for determining the number of repeating units in a sugar are also known in the art. For example, the number of repeating units (or "n" in the formula) can be calculated by dividing the molecular weight of the polysaccharide (the molecular weight without the core sugar or KDO residue) by the molecular weight of the repeating unit (i.e., the molecular weight of the structure in the corresponding formula, such as shown in Table 1, which can theoretically be calculated as the sum of the molecular weights of each monosaccharide in the formula). The molecular weight of each monosaccharide in the formula is known in the art. For example, the molecular weight of the repeating unit of formula O25b is about 862Da. For example, the molecular weight of the repeating unit of formula O1a is about 845Da. For example, the molecular weight of the repeating unit of formula O2 is about 829Da. For example, the molecular weight of the repeating unit of formula O6 is about 893Da. When determining the number of repeating units in the conjugate, the molecular weight of the carrier protein and the protein: polysaccharide ratio are taken into account in the calculation. As defined herein, "n" refers to the number of repeating units in the polysaccharide molecule (represented in brackets in Table 1). As known in the art, in biological macromolecules, the repeating structure can be interspersed with regions of imperfect repetition, such as missing branches. Furthermore, it is known in the art that polysaccharides isolated and purified from natural sources such as bacteria may be heterogeneous in size and branching. In this case, n may represent the average or median value of n of molecules in the population.

[0588] In one embodiment, the O-polysaccharide has at least one O-antigen repeating unit added compared to the corresponding wild-type O-polysaccharide. The repeating units of the O-antigen are shown in Table 1 and Table 1a. In one embodiment, the O-polysaccharide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more total repeating units. Preferably, the saccharide has a total of at least 3 and up to 80 repeating units. In another embodiment, the O-polysaccharide is increased by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more repeating units. In one embodiment, the saccharide comprises an O-antigen wherein n in any O-antigen formula (e.g., the formula shown in Table 1) is an integer of at least 1, 2, 3, 4, 5, 10, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 and at most 200, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51 or 50.Any minimum value and any maximum value can be combined to define a range. Exemplary ranges include, for example, at least 1 to at most 1000, at least 10 to at most 500, and at least 20 to at most 80, preferably at most 90. In a preferred embodiment, n is at least 31 to at most 90. In a preferred embodiment, n is 40 to 90, more preferably 60 to 85.

[0589] In one embodiment, the saccharide comprises an O-antigen, wherein n of any of the O-antigen formulas is at least 1 and at most 200. In one embodiment, n of any of the O-antigen formulas is at least 5 and at most 200. In one embodiment, n of any of the O-antigen formulas is at least 10 and at most 200. In one embodiment, n of any of the O-antigen formulas is at least 25 and at most 200. In one embodiment, n of any of the O-antigen formulas is at least 50 and at most 200. In one embodiment, n of any of the O-antigen formulas is at least 75 and at most 200. In one embodiment, n of any of the O-antigen formulas is at least 100 and at most 200. In one embodiment, n of any of the O-antigen formulas is at least 125 and at most 200. In one embodiment, n of any of the O-antigen formulas is at least 150 and at most 200. In one embodiment, n of any of the O-antigen formulas is at least 175 and at most 200. In one embodiment, n of any of the O-antigen formulas is at least 1 and at most 100. In one embodiment, n of any of the O-antigen formulas is at least 5 and at most 100. In one embodiment, n of any of the O-antigen formulas is at least 10 and at most 100. In one embodiment, n of any of the O-antigen formulas is at least 25 and at most 100. In one embodiment, n of any of the O-antigen formulas is at least 50 and at most 100. In one embodiment, n of any of the O-antigen formulas is at least 75 and at most 100. In one embodiment, n of any of the O-antigen formulas is at least 1 and at most 75. In one embodiment, n of any of the O-antigen formulas is at least 5 and at most 75. In one embodiment, n of any of the O-antigen formulas is at least 10 and at most 75. In one embodiment, n of any of the O-antigen formulas is at least 20 and at most 75. In one embodiment, n of any of the O-antigen formulas is at least 25 and at most 75. In one embodiment, n of any of the O-antigen formulas is at least 30 and at most 75. In one embodiment, n of any of the O-antigen formulas is at least 40 and at most 75. In one embodiment, n of any of the O-antigen formulas is at least 50 and at most 75. In one embodiment, n of any of the O-antigen formulas is at least 30 and at most 90. In one embodiment, n of any of the O-antigen formulas is at least 35 and at most 85. In one embodiment, n of any of the O-antigen formulas is at least 35 and at most 75. In one embodiment, n of any of the O-antigen formulas is at least 35 and at most 70. In one embodiment, n of any of the O-antigen formulas is at least 35 and at most 60. In one embodiment, n of any of the O-antigen formulas is at least 35 and at most 50. In one embodiment, n of any of the O-antigen formulas is at least 35 and at most 49. In one embodiment, n of any of the O-antigen formulas is at least 35 and at most 48.In one embodiment, n of any of the O-antigen formulas is at least 35 and at most 47. In one embodiment, n of any of the O-antigen formulas is at least 35 and at most 46. In one embodiment, n of any of the O-antigen formulas is at least 36 and at most 45. In one embodiment, n of any of the O-antigen formulas is at least 37 and at most 44. In one embodiment, n of any of the O-antigen formulas is at least 38 and at most 43. In one embodiment, n of any of the O-antigen formulas is at least 39 and at most 42. In one embodiment, n of any of the O-antigen formulas is at least 39 and at most 41.

[0590] 84, 85, 86, 87, 88, 89, or 90, most preferably 40. In another embodiment, n is at least 35 and up to 60. For example, in one embodiment, n is any one of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60, preferably 50. In another preferred embodiment, n is at least 55 to at most 75. For example, in one embodiment, n is 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 or 69, most preferably 60.

[0591] The carbohydrate structure can be determined by methods and tools known in the art, such as NMR including 1D, 1H and / or 13C, 2D TOCSY, DQF-COSY, NOESY and / or HMQC.

[0592] In some embodiments, the purified polysaccharide prior to conjugation has a molecular weight between 5 kDa and 400 kDa. In other such embodiments, the saccharide has a molecular weight between 10 kDa and 400 kDa, between 5 kDa and 400 kDa, between 5 kDa and 300 kDa, between 5 kDa and 200 kDa, between 5 kDa and 150 kDa, between 10 kDa and 100 kDa, between 10 kDa and 75 kDa, between 10 kDa and 60 kDa, between 10 kDa and 40 kDa, between 10 kDa and 100 kDa, between 10 kDa and 20 kDa, between 15 kDa and 150 kDa, between 12 kDa and 120 kDa, between 12 kDa and 75 kDa, between 12 kDa and 50 kDa, between 12 and 60 kDa, between 35 kDa and 75 kDa, between 40 kDa and 60 kDa, between 35 kDa and 60 kDa, between 20 kDa and 60 kDa, between 12 kDa and 20 kDa, or between 20 kDa and 50 kDa. In other embodiments, the polysaccharide has a molecular weight of between 7 kDa to 15 kDa, 8 kDa to 16 kDa, 9 kDa to 25 kDa, 10 kDa to 100, 10 kDa to 60 kDa, 10 kDa to 70 kDa, 10 kDa to 160 kDa, 15 kDa to 600 kDa, 20 kDa to 1000 kDa, 20 kDa to 600 kDa, 20 kDa to 400 kDa, 30 kDa to 1,000 KDa, 30 kDa to 60 kDa, 30 kDa to 50 kDa, or 5 kDa to 60 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0593] As used herein, the term "molecular weight" of a polysaccharide or carrier protein-polysaccharide conjugate refers to the molecular weight calculated by size exclusion chromatography (SEC) coupled with a multi-angle laser light scattering detector (MALLS).

[0594] In normal purification procedures, the size of the polysaccharide may be slightly reduced. In addition, as described herein, the polysaccharide may be subjected to sizing techniques prior to conjugation. Mechanical or chemical sizing may be employed. Chemical hydrolysis may be performed using acetic acid. Mechanical sizing may be performed using high pressure homogenization shearing. The above molecular weight ranges refer to purified polysaccharides prior to conjugation (e.g., prior to activation).

[0595] Table 1: Escherichia coli serogroups / serotypes and O-unit fractions

[0596]

[0597]

[0598]

[0599]

[0600]

[0601]

[0602]

[0603]

[0604]

[0605] β-D-6dmanHep2Ac is 2-O-acetyl-6-deoxy-β-D-manno-heptopyranosyl.

[0606] β-D-Xulf is β-D-threopentofuranosyl.

[0607] Core Oligosaccharides

[0608] The core oligosaccharide is located between lipid A and the outer region of the O antigen in wild-type E. coli LPS. More specifically, the core oligosaccharide is part of a polysaccharide that contains a bond between the O-antigen and lipid A in wild-type E. coli. The bond contains a ketoside bond between the hemiketal functional group of the innermost 3-deoxy-d-manno-octano-2-ketosonic acid (KDO) residue and the hydroxyl group of the GlcNAc residue of lipid A. The core oligosaccharide region shows a high degree of similarity in wild-type E. coli strains. It usually contains a limited number of sugars. The core oligosaccharide contains an inner core region and an outer core region.

[0609] More specifically, the inner core is mainly composed of L-glycerol-D-mannose-heptose (heptose) and KDO residues. The inner core is highly conserved. The KDO residues include the following formula KDO:

[0610]

[0611] The outer region of the core oligosaccharide exhibits more variation than the inner core region, and differences in this region distinguish five chemotypes in E. coli: R1, R2, R3, R4, and K-12. HepII is the last residue in the inner core oligosaccharide. Although all outer core oligosaccharides share a structural theme with a (hexose)3 carbohydrate backbone and two side chain residues, the order of the hexoses in the backbone and the nature, position, and linkage of the side chain residues can vary. The structures of the R1 and R4 outer core oligosaccharides are highly similar, differing only in a single β-linked residue.

[0612] The core oligosaccharides of wild-type Escherichia coli are classified in the art into five different chemical types according to the structures of the distal oligosaccharides: Escherichia coli R1, Escherichia coli R2, Escherichia coli R3, Escherichia coli R4 and Escherichia coli K12.

[0613] In a preferred embodiment, the composition described herein comprises a glycoconjugate, wherein the O-polysaccharide comprises a core oligosaccharide that binds to an O-antigen. In one embodiment, the composition induces an immune response against at least any one of the core E. coli chemical types E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12. In another embodiment, the composition induces an immune response against at least two core E. coli chemical types. In another embodiment, the composition induces an immune response against at least three core E. coli chemical types. In another embodiment, the composition induces an immune response against at least four core E. coli chemical types. In another embodiment, the composition induces an immune response against all five core E. coli chemical types.

[0614] In another preferred embodiment, the composition described herein comprises a glycoconjugate, wherein the O-polysaccharide does not comprise a core oligosaccharide that binds to the O-antigen. In one embodiment, the composition induces an immune response against at least any one of the core E. coli chemotypes E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12, even though the glycoconjugate has an O-polysaccharide that does not comprise a core oligosaccharide.

[0615] E. coli serotypes can be characterized according to one of five chemical types. Table 2 lists exemplary serotypes characterized according to chemical type. The serotypes in bold represent the serotypes most commonly associated with the specified core chemical type. Therefore, in a preferred embodiment, the composition induces an immune response against at least any one of the core E. coli chemical types E. coli R1, E. coli R2, E. coli R3, E. coli R4 and E. coli K12, which includes an immune response against any one of the corresponding E. coli serotypes.

[0616] Table 2: Core chemotypes and related E. coli serotypes

[0617]

[0618] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R1 chemotype, for example, a saccharide selected from formula O25a, O6, O2, O1, O75, O4, O16, O8, O18, O9, O13, O20, O21, O91, and O163, wherein n is 1 to 100. In some embodiments, the saccharide in the composition further comprises an E. coli R1 core portion.

[0619] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R1 chemotype, for example, a saccharide selected from formula O25a, O6, O2, O1, O75, O4, O16, O18, O13, O20, O21, O91, and O163, wherein n is 1 to 100, preferably 31 to 100, more preferably 35 to 90, and most preferably 35 to 65. In some embodiments, the saccharide in the composition further comprises an E. coli R1 core portion in the saccharide.

[0620] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R2 chemotype, for example, a saccharide selected from formula O21, formula O44, formula O11, formula O89, formula O162, and formula O9, wherein n is 1 to 100, preferably 31 to 100, more preferably 35 to 90, and most preferably 35 to 65. In some embodiments, the saccharide in the composition further comprises an E. coli R2 core portion.

[0621] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R3 chemotype, for example, a saccharide selected from formula O25b, O15, O153, O21, O17, O11, O159, O22, O86, and O93, wherein n is 1 to 100, preferably 31 to 100, more preferably 35 to 90, and most preferably 35 to 65. In some embodiments, the saccharide in the composition further comprises an E. coli R3 core portion.

[0622] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R4 chemotype, for example, a saccharide selected from formula O2, O1, O86, O7, O102, O160, and O166, wherein n is 1 to 100, preferably 31 to 100, more preferably 35 to 90, and most preferably 35 to 65. In some embodiments, the saccharide in the composition further comprises an E. coli R4 core portion.

[0623] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having a K-12 chemotype (e.g., selected from a saccharide having formula O25b and a saccharide having formula O16), wherein n is 1 to 1000, preferably 31 to 100, more preferably 35 to 90, and most preferably 35 to 65. In some embodiments, the saccharide in the composition further comprises an E. coli K-12 core portion.

[0624] In some embodiments, the carbohydrate comprises a core carbohydrate. Thus, in one embodiment, the O-polysaccharide further comprises an E. coli R1 core portion. In another embodiment, the O-polysaccharide further comprises an E. coli R2 core portion. In another embodiment, the O-polysaccharide further comprises an E. coli R3 core portion. In another embodiment, the O-polysaccharide further comprises an E. coli R4 core portion. In another embodiment, the O-polysaccharide further comprises an E. coli K12 core portion.

[0625] In some embodiments, the carbohydrate does not comprise a core carbohydrate. Thus, in one embodiment, the O-polysaccharide does not comprise an E. coli R1 core portion. In another embodiment, the O-polysaccharide does not comprise an E. coli R2 core portion. In another embodiment, the O-polysaccharide does not comprise an E. coli R3 core portion. In another embodiment, the O-polysaccharide does not comprise an E. coli R4 core portion. In another embodiment, the O-polysaccharide does not comprise an E. coli K12 core portion.

[0626] Carbohydrates and / or polypeptides or fragments thereof derived from Klebsiella pneumoniae

[0627] Klebsiella pneumoniae is a Gram-negative pathogen known to cause urinary tract infections, bacteremia, and sepsis. In one aspect, any composition disclosed herein may further comprise at least one carbohydrate that is or is derived from at least one Klebsiella pneumoniae serotype selected from O1 (and d-Gal-III variants), O2 (and d-Gal-III variants), O2ac, O3, O4, O5, O7, O8, and O12. In a preferred embodiment, any composition disclosed herein may further comprise a polypeptide derived from Klebsiella pneumoniae, the polypeptide being selected from a polypeptide derived from a Klebsiella pneumoniae type I pilus protein or an immunogenic fragment thereof; and a polypeptide derived from a Klebsiella pneumoniae type III pilus protein or an immunogenic fragment thereof.

[0628] As is known in the art, Klebsiella pneumoniae O1 and O2 antigens contain homopolymer galactose units (or galactans). Klebsiella pneumoniae O1 and O2 antigens each contain D-galactan I units (sometimes referred to as O2a repeating units), but the O1 antigen differs in that the O1 antigen has a D-galactan II cap structure. D-galactan III (d-Gal-III) is a variant of D-galactan I. In some embodiments, saccharides derived from Klebsiella pneumoniae O1 comprise repeating units of [→3)-β-D-Galf-(1→3)-α-D-Galp-(1→]. In some embodiments, saccharides derived from Klebsiella pneumoniae O1 comprise repeating units of [→3)-α-D-Galp-(1→3)-β-D-Galp-(1→]. In some embodiments, saccharides derived from Klebsiella pneumoniae O1 comprise repeating units of [→3)-β-D-Gal In some embodiments, the saccharide derived from Klebsiella pneumoniae O1 comprises a repeating unit of f-(1→3)-α-D-Galp-(1→] and a repeating unit of [→3)-α-D-Galp-(1→3)-β-D-Galp-(1→]. In some embodiments, the saccharide derived from Klebsiella pneumoniae O1 comprises a repeating unit of →3)-β-D-Galf-(1→3)-[α-D-Galp-(1→4)]-α-D-Galp-(1→] (referred to as a D-Gal-III repeating unit).

[0629] In some embodiments, saccharides derived from Klebsiella pneumoniae O2 comprise repeating units of [→3)-α-D-Galp-(1→3)-β-D-Galf-(1→] (which may be an element of the Klebsiella pneumoniae serotype O2a antigen). In some embodiments, saccharides derived from Klebsiella pneumoniae O2 comprise repeating units of [→3)-β-D-GlcpNAc-(1→5)-β-D-Galf-(1→] (which may be an element of the Klebsiella pneumoniae serotype O2c antigen). In some embodiments, saccharides derived from Klebsiella pneumoniae O2 comprise repeating units of O2a modified by the addition of a (1→4)-linked Galp residue to the side chain (which may be an element of the Klebsiella pneumoniae O2afg antigen). In some embodiments, saccharides derived from Klebsiella pneumoniae O2 comprise repeating units of O2a modified by the addition of a (1→2)-linked Galp residue to the side chain (which may be an element of the Klebsiella pneumoniae O2aeh antigen).

[0630] Without being bound by mechanism or theory, it is disclosed in the art that the O-antigen polysaccharide structures of K. pneumoniae serotypes O3 and O5 are identical to those of E. coli serotypes O9a (Formula O9a) and O8 (Formula O8), respectively.

[0631] In some embodiments, saccharides derived from Klebsiella pneumoniae O4 comprise repeating units of [→4)-α-D-Galp-(1→2)-β-D-Ribf-(1→)]. In some embodiments, saccharides derived from Klebsiella pneumoniae O7 comprise repeating units of [→2-aL-Rhap-(1→2)-β-D-Ribf-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→]. In some embodiments, saccharides derived from Klebsiella pneumoniae serotype O8 comprise the same repeating unit structure as Klebsiella pneumoniae O2a, but are non-stoichiometrically O-acetylated. In some embodiments, saccharides derived from Klebsiella pneumoniae serotype O12 comprise repeating units of [α-Rhap-(1→3)-β-GlcpNAc] disaccharide repeating units.

[0632] Table 1a. Klebsiella pneumoniae serogroups / serotypes and O-unit fractions

[0633]

[0634]

[0635] As used herein, the term "about" refers to within a statistically significant range of values, such as a specified concentration range, time range, molecular weight, temperature or pH. The range can be within an order of magnitude of a given value or range, typically within 20%, more typically within 10%, and even more typically within 5% or 1%. Sometimes, the range can be within the typical experimental error of a standard method for measuring and / or determining a given value or range. The permissible variations encompassed by the term "about" will depend on the specific system under study, and can be readily understood by a person of ordinary skill in the art. Whenever a range is listed in this application, each number within the range is also considered to be an embodiment of the present disclosure.

[0636] The terms “comprising / comprise / comprises” herein are intended by the inventor to be optionally replaced in each instance with the terms “consisting essentially of / consistessentially of / consists essentially of”, “consisting of / consistof / consists of”, respectively.

[0637] An "immunogenic amount," "immunologically effective amount," "therapeutically effective amount," "prophylactically effective amount," or "dose," each used interchangeably herein, generally refers to the amount of an antigen or immunogenic composition sufficient to elicit an immune response, either cellular (T cell) or humoral (B cell or antibody) response, or both, as measured by standard assays known to those of ordinary skill in the art.

[0638] Any integer within any range in this document is contemplated as an embodiment of the present disclosure.

[0639] All references or patent applications cited in this patent specification are incorporated herein by reference.

[0640] The present invention is illustrated in the accompanying examples. Unless otherwise described in detail, the following examples are carried out using standard techniques, which are well known and routine to those skilled in the art. The examples are illustrative, but not limiting, of the present invention.

[0641] Examples

[0642] Example 1: Escherichia coli and Salmonella enterica strains

[0643] Clinical strains and derivatives are listed in Table 3. Additional reference strains include: O25K5H1, a clinical serotype O25a strain; and Salmonella enterica serotype Typhimurium strain LT2.

[0644] A gene knockout in an E. coli strain that deleted the targeted open reading frame but left a short scar sequence was constructed.

[0645] For simplicity, the hydrolyzed O-antigen chains and core sugars are subsequently denoted as O-polysaccharide (OPS).

[0646]

[0647]

[0648] Example 2: Oligonucleotide primers for cloning of WZZB, FEPE and O-antigen gene clusters

[0649] Table 4. Oligonucleotide primers

[0650]

[0651] Example 3: Plasmid

[0652] Plasmid vectors and subclones are listed in Table 5. PCR fragments containing the wzzB and fepE genes of various E. coli and Salmonella were amplified from purified genomic DNA and subcloned into Invitrogen In the high copy number plasmid provided in the Blunt cloning kit. This plasmid is based on the pUC replicon. Primers P3 and P4 are used to amplify the E. coli wzzB gene with a natural promoter and are designed to bind to the regions of the proximal and distal genes encoding UDP-glucose-6-dehydrogenase and phosphoribosyl adenine nucleotide hydrolase, respectively (annotated in Genbank MG1655NC_000913.3). A PCR fragment containing the Salmonella fepE gene and promoter is amplified using the primers described previously. Similar E. coli fepE primers are designed based on available Genbank genome sequences or internally generated full genome data (in the case of GAR2401 and O25K5H1). Low copy number plasmid pBAD33 is used to express O-antigen biosynthesis genes under the control of the arabinose promoter. The plasmid was first modified to facilitate cloning (by the Gibson method) of a long PCR fragment amplified using universal primers homologous to the 5' promoter and 3' 6-phosphogluconate dehydrogenase (gnd) gene Table 5.

[0653] Table 5. Plasmids

[0654]

[0655] Example 4: O-antigen purification

[0656] The fermentation broth was treated with acetic acid to a final concentration of 1%-2% (final pH 4.1). OAg extraction and defatting was achieved by heating the acid-treated broth to 100°C for 2 hours. At the end of the acid hydrolysis, the batch was cooled to ambient temperature and 14% NH 4 OH to a final pH of 6.1. The neutralized culture broth was centrifuged and the centrifuge was collected. CaCl 2 The resulting slurry was incubated at room temperature for 30 minutes.

[0657] Solids were removed by centrifugation, and the centrate was concentrated 12-fold using a 10 kDa membrane and then diafiltered twice against water. The retentate containing OAg was then purified using a carbon filter. The carbon filtrate was diluted 1:1 (v / v) with 4.0 M ammonium sulfate. The final ammonium sulfate concentration was 2 M. The ammonium sulfate treated carbon filtrate was further purified using a membrane with 2 M ammonium sulfate as the operating buffer. OAg was collected in the downstream. For long OAg, the HIC filtrate was concentrated and the buffer was then exchanged with water (20 diafiltration volumes) using a 5 kDa membrane. For short (native) OAg polysaccharides, the MWCO was further reduced to increase yield.

[0658] In another embodiment, solids are removed by centrifugation and the centrate is concentrated 12 times using a 10 kDa membrane and then diafiltered twice against water or 20-25 mM Tris buffer containing 20-25 mM NaCl pH 7.2-7. The retentate containing OAg is then purified using a carbon filter. The carbon filtrate is further purified by ion exchange (IEX) membrane chromatography. The IEX filtrate is then diluted with 4.0 M ammonium sulfate 1: 1 (v / v). The final ammonium sulfate concentration is 2 M. The ammonium sulfate treated IEX filtrate is further purified using a HIC membrane with 2 M ammonium sulfate as the operating buffer. OAg is collected in the downstream. The HIC filtrate is concentrated and the buffer is then exchanged with water (20 diafiltration volumes) using a 5 kDa membrane.

[0659] Background of Examples 5-17: The examples described here demonstrate a platform-based approach for purification of all serotypes of O-antigen polysaccharides (also referred to as O-antigens or O-Ags) that may contain internal / external oligosaccharides.

[0660] The purification methods described herein are applicable to both short-chain and long-chain O-Ag polysaccharides. Most of the examples given herein are for long-chain O-Ag, except for Examples 10 and 11, which are short-chain O-Ag of E. coli serotypes O8 and O9, respectively.

[0661] Example 5: Method for purifying Escherichia coli O-antigen polysaccharide

[0662] 1. Release of O-antigen

[0663] The process begins with acid hydrolysis after fermentation is complete to release O-Ag from lipopolysaccharide (LPS). This is achieved by treating a crude suspension of serotype O25b cell culture with acetic acid to a final concentration of 1.0% (v / v) to a pH of about 4.0. The acidic broth is then heated to a temperature of 100°C and incubated for 2.0 hours. After product release, the batch is cooled to ambient temperature 20-30°C.

[0664] To further optimize the release conditions of O25b O-Ag, a design of experiments (DOE) was established to examine the effects of pH, temperature, and hold time on %KDO, concentration, molecular weight (MW), and O-acetate. The factors examined in the DOE study are shown in Table 1-1. Note that for this study, the initial concentration, upper limit of KDO, and upper limit of O-acetate were set to 3.5 mg / mL, 2%, and 1.0 mM, respectively.

[0665] Table 1-1: DOE factors tested in the O25b acid hydrolysis study

[0666] factor scope pH 3.0-5.0 Temperature(℃) 80-100 Holding time (hours) 1.0-4.0

[0667] The predicted responses for concentration, %KDO, and O-acetate were evaluated at the 1.0, 2.0, 2.5, and 4.0 hour time points. The molecular weight was stable at approximately 50-54 kDa under all conditions, except for the temperature condition of 80°C, where the MW was larger. This may be due to incomplete product release, in which case O-Ag may bind to cellular components or other nonspecific surface polysaccharides. Therefore, the DOE model was unable to provide a predicted response for MW under these conditions. At the 4.0 hour time point, no %KDO was in range.

[0668] Based on this DOE result, the conditions for acid hydrolysis were reset to pH 3.8±0.1, temperature 95±5°C, and a hold time of 2.0 hours. In subsequent examples, these release conditions were used for all other serotypes of O-Ag polysaccharides.

[0669] 2. Flocculation

[0670] The main purpose of this step is to precipitate cell debris, host cell proteins, and nucleic acids from the culture broth containing the released product. It also improves the efficiency of the downstream clarification unit operation. The acidified culture broth after the release of the step 1 product is treated with 10% alum solution to a final concentration of 2% (w / v) and the pH is further adjusted to 3.2 using sulfuric acid. The flocculated slurry is incubated at ambient temperature for 1.0 hour and then centrifuged at 12,000-14,000g for 30 minutes. The supernatant is then filtered with a 0.2-μm filter or another suitable depth filter to remove any small particles that may have jumped into the solution. The depth filtrate undergoes preliminary purification of UFDF-1.

[0671] Alternatively, the acidified culture broth is neutralized to a pH of 6.0-7.0. The neutralized filtrate is centrifuged at 12,000 g for 30 minutes. The neutralized supernatant is filtered through a 0.2-μm filter. The neutralized filtrate can be stored at 4°C for at least one week without any adverse effect on product quality. When the batch is ready for purification, the neutralized filtrate is subjected to the flocculation process described in the previous paragraph. Unless otherwise stated, the subsequent purification steps described in this example use this flocculation method.

[0672] The SEC-HPLC chromatograms of the neutralization filtrate containing the product and the depth filtrate after flocculation were compared. From the refractive index (RI) and UV 280 detection results, the flocculation step removed a large number of impurities inherited from the fermentation medium.

[0673] 3. Ultrafiltration / diafiltration-(UFDF-1)

[0674] The depth filtrate from step 2 above was further purified by ultrafiltration and diafiltration (UFDF) using a 10-kDa Sartocon Hydrosart membrane. The amount of depth filtrate processed was typically2 Membrane area 20-30 liters. The purpose of this operation is: (i) to reduce the volume by concentrating the solution 10-20 times, and (ii) to exchange the buffer by replacing the fermentation medium with the required buffer by diafiltration. The buffer used in this step is 20mM citrate / 0.1M NaCl pH 6.0, followed by a second buffer of 20mM Tris / 20mM NaCl pH 7.2. The diafiltration volume number for both diafiltration steps is 10. The retentate from UFDF is collected and analyzed. The conductivity and UV curves during the UFDF run show that during the first diafiltration, most of the small MW and UV-related impurities are removed, and the UV signal of the permeate is significantly reduced to prove this. The SEC-HPLC chromatogram comparison of the depth filtrate and the UFDF-1 retentate was analyzed for RI and UV detection.

[0675] 4. Carbon Filtration

[0676] This unit operation reduces the content of host cell impurities such as proteins and nucleic acids as well as colored impurities (see WO2008118752). When using 3M R32SP carbon filters, each m 2 The carbon filter area can be loaded with approximately 150 g of O-Ag from the retentate of UFDF-1. The carbon filter is first flushed with water and then with diafiltration buffer for every m 2 The filtration area is about 20 liters of buffer. Then, at 50 LMH (liter / m 2 The retentate from UFDF-1 was filtered in single pass mode at a flow rate of 1.5 % / hr. The filter was then rinsed with buffer and the filtrate containing the product, including the rinse, was collected as the carbon filtrate.

[0677] SEC-HPLC chromatograms of the UFDF retentate and carbon filtrate showed that RI and UV280 related impurities were removed and the carbon filtrate became visually colorless.

[0678] 5. IEX membrane chromatography

[0679] This step was initially developed for serotype O2 and O6 O-antigens to remove non-specific negatively charged impurities (see Examples 6 and 15). Therefore, impurities from non-serotype specific extracellular or intracellular polysaccharides can be removed by exploring the electrostatic interaction properties of these molecules using ion exchange (IEX) membrane chromatography.

[0680] The IEX membrane used here is the NatriFlo membrane cassette from Millipure. Alternatively, the Sartobind Q membrane from Sartorius Stedim can also be used. Unless otherwise stated, all examples described here use NatriFlo membrane (or HD-Q hereinafter) for IEX membrane chromatography.

[0681] The membrane is first balanced with 20mM Tris / 20mM NaCl pH 7.2, typically 20-30 membrane volumes (MV). The carbon filtrate from the previous step is pumped through the membrane at a flow rate of 30-40mL / min, containing approximately 200-250mg O-Ag per mL MV of carbon filtrate. The effluent or filtrate containing the product is collected. The membrane is rinsed with the equilibration buffer and then washed with a high salt buffer of 20mM Tris / 1.0M NaCl pH 7.2. Conductivity and UV280 curves of the IEX membrane chromatography run, in which the UV signal shows a peak during the high salt wash, indicating the presence of unknown negatively charged impurities in the carbon filtrate.

[0682] SEC-HPLC chromatograms of the charcoal filtrate, IEX filtrate, and high salt wash effluent showed that the high salt wash chromatogram showed a small peak at the same retention time as the product peak. This indicated that this unknown substance had a stronger ionic strength than O25b O-Ag.

[0683] 6. Hydrophobic Interaction Chromatography (HIC)

[0684] This unit operation removes any impurities with hydrophobic properties, such as residual lipid A left over from the acid hydrolysis step. Sartobind Phenyl 150-mL membrane is used for the HIC step. The charcoal filtrate from step 4 is treated with 4.0 M ammonium sulfate (AS) solution to a final concentration of 2.0 M. The phenyl membrane is first equilibrated with 2.0 M ammonium sulfate (AS) operating buffer. The AS-treated charcoal filtrate is passed through the HIC membrane at a flow rate of 40 mL / min. The HIC membrane is then rinsed with operating buffer and then washed with water. The effluent is collected as the HIC filtrate along with the buffer rinse, and the water wash is also collected for analysis. Alternatively, this HIC filtration step can also be performed on the IEX filtrate from step 4.

[0685] The AKTA Avant chromatogram used for the HIC purification run was analyzed. The product was in the flow through effluent and the peak shown in the water wash was a non-specific hydroph...

Claims

1. A method for purifying sugars derived from bacteria from a solution comprising sugars and contaminants after fermentation, wherein the method comprises the following steps: a) Acid hydrolysis; b) first ultrafiltration / diafiltration - (UFDF-1); b) Carbon filtration; c) chromatography; and d) Second ultrafiltration / diafiltration - (UFDF-2).

2. The method according to claim 1, further comprising a flocculation step after the acid hydrolysis in step (a).

3. The method according to claim 1 or 2, wherein the chromatography of step (c) comprises IEX membrane chromatography or hydrophobic interaction chromatography (HIC) or both.

4. The method according to any one of claims 1 to 3, wherein the bacteria are Gram-positive bacteria.

5. The method according to claim 4, wherein the bacterium is any one of the genus Streptococcus, Staphylococcus, Enterococcus, Bacillus, Corynebacterium, Listeria, Erysipelothrix or Clostridium.

6. The method of claim 5, wherein the bacterium is any one of Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Group C and Group G Streptococci, or Staphylococcus aureus.

7. The method according to any one of claims 1 to 3, wherein the bacteria are Gram-negative bacteria.

8. The method according to claim 7, wherein the bacterium is any one of the genus Haemophilus, Neisseria, Escherichia or Klebsiella.

9. The method of claim 8, wherein the bacterium is Haemophilus influenzae, Neisseria meningitidis, Escherichia coli or Klebsiella pneumoniae.

10. The method according to claim 9, wherein the bacteria is Escherichia coli comprising a saccharide having a structure selected from any one of the following: formula O1, formula O1A, formula O1B, formula O1C, formula O2, formula O3, formula O4, formula O4:K52, formula O4:K6, formula O5, formula O5ab, formula O5ac, formula O6, formula O6:K2; K13; K15, formula O6:K54, formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18, formula O18A, formula O18ac, formula O18A1, formula O18B, formula O18B1, formula O19, formula O20, formula O21, formula O22, formula O23, formula O24, formula O25 23. O23a, O24, O25, O25a, O25b, O26, O27, O28, O29, O30, O32, O33, O34, O35, O36, O37, O38, O39, O40, O41, O42, O43, O44, O45 5. Formula O45, Formula O45rel, Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula 62D1, Formula O63, Formula O64, Formula O65, Formula O66, Formula O68 , O69, O70, O71, O73, O74, O75, O76, O77, O78, O79, O80, O81, O82, O83, O84, O85, O86, O87, O88, O89, O90, O91, O92, O93 3. Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formula O115, Formula O116 16. Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138、O139、O140、O141、O142、O143、O144、O145、O146、O147、O148、O149、O150、O151、O152、O153、O154、O155、O156、O157、O158、Formula O159, formula O160, formula O161, formula O162, formula O163, formula O164, formula O165, formula O166, formula O167, formula O168, formula O169, formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186 or formula O187.

11. The method of claim 9, wherein the bacterium is Klebsiella pneumoniae comprising a carbohydrate having a structure selected from any one of the following: formula K.O1.1, formula K.O1.2, formula K.O1.3, formula K.O1.4, formula K.O2.1, formula K.O2.2, formula K.O2.3, formula K.O2.4, formula K.O3, formula K.O4, formula K.O5, formula K.O7, formula K.O12 or formula K.O8.

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