Sugar refining

Abstract

To provide a robust and effective purification process that can be used in large-scale production of bacterial polysaccharides after fermentation.SOLUTION: This invention relates to a method for purifying bacterial polysaccharides, particularly removing impurities from cell lysates of bacteria producing polysaccharides.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Sequence Listing Reference This application was filed electronically via EFS-Web and contains a sequence listing that has been submitted electronically in .txt format. The .txt file contains a sequence listing entitled "PC72592_ST25.txt," created on January 29, 2021, and having a size of 34 KB. The sequence listing contained in this .txt file is a part of the present specification and is incorporated herein by reference in its entirety.

[0002] Technical Field The present invention relates to a method for purifying bacterial polysaccharides, and in particular for removing impurities from cell lysates of polysaccharide-producing bacteria. [Background technology]

[0003] Bacterial polysaccharides, particularly capsular polysaccharides, are important immunogens found on the surface of bacteria involved in a variety of bacterial diseases. This has made them important components in vaccine design. They have proven useful in eliciting immune responses, especially when linked to carrier proteins.

[0004] 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).

[0005] Typically, bacterial polysaccharides are produced using batch, fed-batch or continuous culture in complex media. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2007 / 052168 [Patent Document 2] WO2009 / 081276 [Patent Document 3] WO2010151544 [Patent Document 4] WO2011 / 051917 [Patent Document 5] WO2007084856 [Patent Document 6] WO2008 / 118752, page 13, line 14 ~ page 14, line 10 [Patent Document 7] WO2006 / 110381 [Patent Document 8] WO2007116028, pages 17-22 [Patent Document 9] WO2015110941 [Patent Document 10] WO2015110940 [Patent Document 11] WO2014097099 [Patent Document 12] WO93 / 15760 [Patent Document 13] WO95 / 08348 [Patent Document 14] WO96 / 129094 [Patent Document 15] WO2011041003 [Patent Document 16] WO2014027302 [Patent Document 17] WO2015121783 [Patent Document 18] WO00 / 10599 [Patent Document 19] U.S. Patent No. 4,673,574 [Patent Document 20] U.S. Patent No. 4,808,700 [Patent Document 21] U.S. Patent No. 4,459,286 [Patent Document 22] U.S. Patent No. 4,709,017 [Patent Document 23] U.S. Patent No. 4,950,740 [Patent Document 24] U.S. Patent No. 5,917,017 [Patent Document 25] U.S. Patent No. 6,455,673 [Patent Document 26] U.S. Patent No. 5,843,711 [Patent Document 27] WO2004 / 081515 [Patent Document 28] WO2006 / 032499 [Patent Document 29] WO00 / 37105 [Patent Document 30] WO00 / 39299 [Patent Document 31] WO01 / 98334 [Patent Document 32] WO03 / 054007 [Patent Document 33] WO2009 / 000826 [Patent Document 34] EP0372501 [Patent Document 35] EP0594610B [Patent Document 36] EP0378881 [Patent Document 37] EP0427347 [Patent Document 38] WO93 / 17712 [Patent Document 39] WO94 / 03208 [Patent Document 40] WO98 / 58668 [Patent Document 41] EP0471177 [Patent Document 42] WO91 / 01146 [Patent Document 43] WO02 / 091998 [Patent Document 44] WO01 / 72337 [Patent Document 45] WO00 / 61761 [Patent Document 46] WO2004 / 083251 [Patent Document 47] WO01 / 98334 [Patent Document 48] WO03 / 054007 [Patent Document 49] EP0594610B [Patent Document 50] U.S. Patent No. 5,614,382 [Patent Document 51] CN103495161 [Patent Document 52] WO00 / 07621 [Patent Document 53] WO99 / 44636 [Patent Document 54] WO00 / 56358 [Patent Document 55] GB-2220221 [Patent Document 56] EP0689454 [Patent Document 57] EP0835318 [Patent Document 58] EP0735898 [Patent Document 59] EP0761231 [Patent Document 60] WO99 / 52549 [Patent Document 61] WO01 / 21207 [Patent Document 62] WO01 / 21152 [Patent Document 63] WO00 / 62800 [Patent Document 64] WO00 / 23105 [Patent Document 65] WO99 / 11241 [Patent Document 66] WO98 / 57659 [Non-patent literature]

[0007] [Non-Patent Document 1] Fattom et al. (1990) Infect Immun. 58(7):2367-74 [Non-patent document 2] Uchida et al. (1973) J. Biol. Chem. 218:3838~3844 [Non-patent document 3] Nicholls and Youle in Genetically Engineered Toxins, Eds: Frankel, Maecel Dekker Inc. (1992) [Non-patent document 4] Kuo et al. (1995) Infect Immun 63:2706-2713 [Non-Patent Document 5] Falugi et al. (2001) Eur J Immunol 31:3816~3824 [Non-patent document 6] Baraldoi et al. (2004) Infect Immun 72:4884-4887 [Non-Patent Document 7] Douglas et al. (1987) J. Bacteriol. 169(11):4967~4971 [Non-patent document 8] Uchida et al. (1971) Nature New Biology 233:8-11 Summary of the Invention [Problem to be solved by the invention]

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

[0009] Thus, to produce substantially purified bacterial saccharides suitable for incorporation into vaccines, a simplified purification process is also needed to reduce the soluble protein levels in bacterial lysates and to eliminate inefficiencies in current purification processes. [Means for solving the problem]

[0010] The present invention provides a method for purifying bacterially derived sugars from a solution containing said sugars and contaminants after fermentation, the method comprising the following steps: (a) acid hydrolysis; (b) first ultrafiltration / diafiltration (UFDF-1); (c) carbon filtration; (d) chromatography; and (e) second ultrafiltration / diafiltration (UFDF-2).

[0011] In one embodiment, the method further comprises a flocculation step after the acid hydrolysis of step (a).

[0012] In another embodiment of the method, the bacterium is a Gram-positive bacterium. In one aspect, the bacterium is any one of Streptococcus, Staphylococcus, Enterococci, Bacillus, Corynebacterium, Listeria, Erysipelothrix, or Clostridium. In a further aspect, the bacterium is any one of Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, group C and G streptococci, or Staphylococcus aureus.

[0013] In another embodiment of the above method, the bacterium is a gram-negative bacterium. In one aspect, the bacterium is any one of Haemophilus, Neisseria, Escherichia, or Klebsiella. In another aspect, the bacterium is Haemophilus influenzae, Neisseria meningitidis, Escherichia coli, or Klebsiella pneumoniae. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 depicts SEC-HPLC chromatograms of Kp O-Ag after release in broth (top) and purified Kp O-Ag (bottom) for the O1V1 variant. [Figure 2] FIG. 1 depicts SEC-HPLC chromatograms of Kp O-Ag after release in broth (top) and purified Kp O-Ag (bottom) for O1V2 variants. [Figure 3] FIG. 1 depicts SEC-HPLC chromatograms of Kp O-Ag after release in broth (top) and purified Kp O-Ag (bottom) for the O2V1 variant. [Figure 4] FIG. 1 depicts SEC-HPLC chromatograms of Kp O-Ag after release in broth (top) and purified Kp O-Ag (bottom) for the O2V2 variant. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention provides a method for purifying bacterially derived sugars from a solution containing said sugars and contaminants after fermentation, the method comprising the following steps: (a) acid hydrolysis; (b) first ultrafiltration / diafiltration (UFDF-1); (c) carbon filtration; (d) chromatography; and (e) second ultrafiltration / diafiltration (UFDF-2).

[0016] In one embodiment, the method further comprises a flocculation step after the acid hydrolysis of step (a).

[0017] In another embodiment of the above method, the chromatography in step (c) comprises IEX membrane chromatography or hydrophobic interaction chromatography (HIC) or both.

[0018] In another embodiment of the method, the bacterium is a Gram-positive bacterium. In one aspect, the bacterium is any one of Streptococcus, Staphylococcus, Enterococci, Bacillus, Corynebacterium, Listeria, Erysipelothrix, or Clostridium. In a further aspect, the bacterium is any one of Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, or group C and G streptococci, or Staphylococcus aureus.

[0019] In another embodiment of the above method, the bacterium is a gram-negative bacterium. In one aspect, the bacterium is any one of Haemophilus, Neisseria, Escherichia, or Klebsiella. In another aspect, the bacterium is Haemophilus influenzae, Neisseria meningitidis, Escherichia coli, or Klebsiella pneumoniae.

[0020] In a further aspect, the bacterium is of the 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, formula O25a, formula O25b, formula O26, formula O27, formula O28, formula O29, formula O30, formula O32, formula O33, formula O34, formula O35, formula O36, formula O37, formula O38, formula O39, formula O40, formula O41, formula O42, formula O43, formula O44, formula O45, 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, formula O69, formula O70, formula 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, 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 0111, formula O112, formula O113, formula O114, formula O115, formula O116, 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, formula O139, formula O140, formula O14A, formula O142, formula O143, formula O144, formula O145, formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula O163, formula O164and an Escherichia coli comprising a saccharide having a structure selected from any one of 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.

[0021] In another aspect, the bacterium is Klebsiella pneumoniae comprising a saccharide having a structure selected from any one of 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.

[0022] 1. Purification process of bacterial polysaccharides 1.1 Starting materials The method of the present invention can be used to purify bacterial polysaccharides from a solution containing said polysaccharides together with contaminants.

[0023] 1.1.1. Bacterial cells The source of the bacterial polysaccharides purified according to the present invention is bacterial cells, particularly pathogenic bacteria.

[0024] Non-limiting examples of Gram-positive bacteria for use according to the present invention are Streptococcus (e.g., S. pneumoniae, S. pyogenes, S. agalactiae or group C and G Streptococci), Staphylococcus (e.g., Staphylococcus aureus), Enterococci, Bacillus, Corynebacterium, Listeria, Erysipelothrix, and Clostridium. Non-limiting examples of Gram-negative bacteria for use with 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).

[0025] In one embodiment, sources of bacterial polysaccharides for use according to the present invention include Aeromonas hydrophila and other spp.; Bacillus anthracis; Bacillus cereus; Botulinum neurotoxin-producing species of Clostridium; Brucella abortus; Brucella melitensis; Brucella suis; Burkholderia mallei (formerly Pseudomonas mallei); Burkholderia pseudomallei (formerly Pseudomonas pseudomallei); Campylobacter jejuni; jejuni; Chlamydia psittaci; Chlamydia trachomatis; Clostridium botulinum; Clostridium difficile; Clostridium perfringens; Coccidioides immitis; Coccidioides posadasii; Cowdria ruminantium (heartwater disease); Coxiella burnetii; Enterococcus faecalis;Enterotoxigenic Escherichia coli (EEC group) such as enterotoxigenic Escherichia coli (ETEC), pathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC)-O157:H7, and enteroinvasive Escherichia coli (EIEC); Ehrlichia spp. such as Ehrlichia chajfeensis; Francisella tularensis; Legionella pneumophila; Liberobacter africanus; Liberobacter asiaticus; Listeria monocytogenes; monocytogenes; miscellaneous enterobacteria 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 shigelloides; Ralstonia solanacearum race 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; Xylella fastidiosa (citrus variegated yellows strain); Yersinia enterocolitica enterocolitica and Yersinia pseudotuberculosis; and Yersinia pestis;

[0026] Polysaccharides desirable for purification can be associated with cellular components such as the cell wall, meaning that the polysaccharide is a component of the cell wall itself and / or is attached to the cell wall, either directly or indirectly through an intermediate molecule, or is a transient coating of the cell wall (e.g., certain bacterial strains exude capsular polysaccharides, also known in the art as "exopolysaccharides").

[0027] 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.

[0028] In one embodiment, the source of the bacterial capsular polysaccharide is Escherichia coli. In a further embodiment, the source of the bacterial capsular polysaccharide is an Escherichia coli portion of the enterotoxigenic Escherichia coli group (EEC group), such as enterotoxigenic Escherichia coli (ETEC), pathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli-O157:H7 (EHEC), or enterocytoinvasive Escherichia coli (EIEC). In one embodiment, the source of the bacterial capsular polysaccharide is uropathogenic Escherichia coli (UPEC).

[0029] In another embodiment, the source of bacterial capsular polysaccharides is an Escherichia coli serotype selected from the group consisting of serotypes O157:H7, O26:H11, O111:H-, and O103:H2. In one embodiment, the source of bacterial capsular polysaccharides is an Escherichia coli serotype selected from the group consisting of serotypes O6:K2:H1 and O18:K1:H7. In one embodiment, the source of bacterial capsular polysaccharides is an Escherichia 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 bacterial capsular polysaccharides is Escherichia coli serotype O104:H4. In one embodiment, the source of bacterial capsular polysaccharides is Escherichia coli serotype O1:K12:H7. In one embodiment, the source of bacterial capsular polysaccharides is Escherichia coli serotype O127:H6. In one embodiment, the source of bacterial capsular polysaccharides is Escherichia coli serotype O139:H28. In one embodiment, the source of bacterial capsular polysaccharides is Escherichia coli serotype O128:H2.

[0030] In another embodiment, the source of bacterial capsular polysaccharides is Neisseria meningitidis. In one embodiment, the source of bacterial capsular polysaccharides is N. meningitidis serogroup A (MenA), N. meningitidis serogroup W135 (MenW135), N. meningitidis serogroup Y (MenY), N. meningitidis serogroup X (MenX), or N. meningitidis serogroup C (MenC). In one embodiment, the source of bacterial capsular polysaccharides is N. meningitidis serogroup A (MenA). In one embodiment, the source of bacterial capsular polysaccharides is N. meningitidis serogroup W135 (MenW135). In one embodiment, the source of bacterial capsular polysaccharides is N. meningitidis serogroup Y (MenY). In one embodiment, the source of bacterial capsular polysaccharides is N. meningitidis serogroup C (MenC). In one embodiment, the source of bacterial capsular polysaccharides is N. meningitidis serogroup X (MenX).

[0031] In a further embodiment, the source of bacterial capsular polysaccharides is Klebsiella pneumoniae. In one embodiment, the source of bacterial capsular polysaccharides 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 O6 (O6), Klebsiella pneumoniae serogroup O7 (O7), Klebsiella pneumoniae serogroup O8 (O8), Klebsiella pneumoniae serogroup O9 (O9), Klebsiella pneumoniae serogroup O10 (O10), Klebsiella pneumoniae serogroup O11 (O11), Klebsiella pneumoniae serogroup O12 (O12), Klebsiella pneumoniae serogroup O13 (O13), Klebsiella pneumoniae serogroup O14 (O14), Klebsiella pneumoniae serogroup O15 (O15), Klebsiella pneumoniae serogroup O16 (O16), Klebsiella pneumoniae serogroup O17 (O17), Klebsiella pneumoniae serogroup O18 (O18), Klebsiella pneumoniae serogroup O19 (O19), Klebsiella pneumoniae serogroup O2ac (O2ac), Klebsiella pneumoniae serogroup O2ac (O2ac), Kleb In one embodiment, the source of bacterial capsular polysaccharide is K. pneumoniae serogroup O4 (O4), K. pneumoniae serogroup O5 (O5), K. pneumoniae serogroup O7 (O7), K. pneumoniae serogroup O8 (O8), or K. pneumoniae serogroup O9 (O9). In one embodiment, the source of bacterial capsular polysaccharide is K. pneumoniae serogroup O1 (O1). In one embodiment, the source of bacterial capsular polysaccharide is K. pneumoniae serogroup O2 (O2). In some embodiments, the source of bacterial capsular polysaccharides is K. pneumoniae serogroup O2ac (O2ac). In some embodiments, the source of bacterial capsular polysaccharides is K. pneumoniae serogroup O3 (O3). In some embodiments, the source of bacterial capsular polysaccharides is K. pneumoniae serogroup O4 (O4). In some embodiments, the source of bacterial capsular polysaccharides is K. pneumoniae serogroup O5 (O5). In some embodiments, the source of bacterial capsular polysaccharides is K. pneumoniae serogroup O7 (O7). In one embodiment, the source of bacterial capsular polysaccharides is K. pneumoniae serogroup O8 (O8). In one embodiment, the source of bacterial capsular polysaccharides is K. pneumoniae serogroup O9 (O9).

[0032] 1.1.2. Bacterial Cell Growth Typically, the polysaccharides are produced by growing the bacteria in a medium (e.g., solid or, preferably, liquid medium), and the bacterial cells are then treated to prepare the polysaccharides.

[0033] Thus, in certain embodiments, the starting material for the methods of the present invention is a bacterial culture, preferably a liquid bacterial culture (eg, a fermentation broth).

[0034] Bacterial cultures are typically obtained by batch, fed-batch, or continuous culture (see, for example, WO2007 / 052168 or WO2009 / 081276). During continuous culture, fresh medium is added to the culture at a fixed rate, and cells and medium are removed at a rate that maintains a constant culture volume.

[0035] A population of organisms is often scaled up from seed vials to seed bottles and passaged through one or more larger seed fermentors until a production-scale fermentation capacity is reached.

[0036] 1.1.3 Pretreatment of bacterial cells to obtain starting material Generally, small amounts of polysaccharides are released into the culture medium during bacterial growth, and therefore the starting material may be the supernatant from a centrifuged bacterial culture, however, typically the starting material will be prepared by treating the bacteria themselves so as to release the polysaccharides.

[0037] Optionally, after cell growth, the bacterial cells are inactivated. This is particularly true when pathogenic bacteria are used. A suitable method for inactivation is, for example, treatment with phenol:ethanol, as described, for example, in Fattom et al. (1990) Infect Immun. 58(7):2367-74. In the following embodiments, the bacterial cells may or may not be pre-inactivated.

[0038] Polysaccharides can be released from bacteria by a variety of methods, including chemical, physical or enzymatic treatment (see, for example, WO2010151544, WO2011 / 051917 or WO2007084856).

[0039] In certain embodiments, the bacterial cells (inactivated or not) are treated in suspension in their original culture medium. The process may therefore begin with the cells in suspension in their original culture medium.

[0040] In another embodiment, the bacterial cells are centrifuged prior to release of the capsular polysaccharides. The process may therefore begin with the cells in the form of a wet cell paste. Alternatively, the cells are processed in dry form. Typically, however, after centrifugation, the bacterial cells are resuspended in an aqueous medium suitable for the next step in the process, such as a buffer or distilled water. The cells may be washed with this medium prior to resuspension.

[0041] In some embodiments, bacterial cells (e.g., in suspension in their original culture medium, in the form of a wet cell paste, in dry form, or resuspended in an aqueous medium after centrifugation) are treated with a lysing agent. A "lysing agent" is any agent that aids in cell wall disruption. In some embodiments, the lysing agent is a detergent. As used herein, the term "detergent" refers to any anionic or cationic detergent capable of inducing lysis of bacterial cells. Representative examples of such detergents for use in the methods of the present invention include sodium deoxycholate (DOC), N-lauryl sarcosine (NLS), sodium chenodeoxycholate, and saponin (see WO 2008 / 118752, page 13, line 14 to page 14, line 10). In one embodiment of the present invention, the lysing agent used to lyse bacterial cells is DOC.

[0042] In some embodiments, 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(oxyethylene)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), N-lauryl sarcosine sodium (NLS), lauryliminodipropionic acid, sodium dodecyl sulfate, chenodeoxycholate, hyodeoxycholate, glycodeoxycholate, taurodeoxycholate, taurochenodeoxycholate, and cholate. In some embodiments, the non-animal-derived dissolving agent is NLS.

[0043] In some embodiments, bacterial cells (e.g., in suspension in their original culture medium, in the form of a wet cell paste, in dry form, or resuspended in an aqueous medium after centrifugation) are enzymatically treated to release polysaccharides. In some embodiments, 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, which act on bacterial peptidoglycan to release capsular saccharides for use with the present invention, but also result in the release of group-specific carbohydrate antigens. In some embodiments, bacterial cells are treated with type II phosphodiesterase (PDE2).

[0044] Optionally, after release of the polysaccharide, the enzyme is inactivated. Suitable methods for inactivation are, for example, heat treatment or acid treatment.

[0045] In some embodiments, bacterial cells (e.g., in suspension in their original culture medium, in the form of a wet cell paste, in dry form, or resuspended in an aqueous medium after centrifugation) are autoclaved to release the polysaccharides.

[0046] In further embodiments, the bacterial cells (e.g., in suspension in their original culture medium, in the form of a wet cell paste, in dry form, or resuspended in an aqueous medium after centrifugation) are chemically treated to release the polysaccharides. In such embodiments, the chemical treatment may be, for example, hydrolysis using a base or an acid (see, e.g., WO2007084856).

[0047] In some embodiments, the chemical treatment of the bacterial cells is base extraction (e.g., using sodium hydroxide). Base extraction can cleave the phosphodiester bond between the capsular saccharide and the peptidoglycan backbone. In some embodiments, 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 base treatment, the reaction mixture can be neutralized. This can be achieved by adding an acid. In some embodiments, after base treatment, the reaction mixture is neutralized with an acid selected from the group consisting of HCl, H3PO4, citric acid, acetic acid, nitrous acid, and sulfuric acid.

[0048] In some embodiments, the chemical treatment of the bacterial cells is an acid treatment (e.g., sulfuric acid). In some embodiments, the acid is selected from the group consisting of HCl, H3PO4, 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 some embodiments, after the acid treatment, the reaction mixture is neutralized with a base selected from the group consisting of NaOH, KOH, LiOH, NaHCO3, Na2CO3, KzCO3, KCN, Et3N, NH3, HzN2H2, NaH, NaOMe, NaOEt, and KOtBu.

[0049] 1.2 Coagulation The method of the present invention includes a flocculation step, and the inventors have found that this process is rapid and simple, resulting in purified polysaccharides that are low in contaminants.

[0050] Thus, in the method of the present invention, the solution obtained by any of the methods in section 1.1 above is treated by flocculation.

[0051] In the present invention, the term "flocculation" refers to the process by which colloids come out of suspension in the form of flocs or flakes due to the addition of a flocculating agent.

[0052] The flocculation step involves adding a "flocculating agent" to a solution containing bacterial polysaccharides along with contaminants. In some embodiments, the contaminants include bacterial cell debris, bacterial cell proteins, and nucleic acids. In some embodiments, the contaminants include bacterial cell proteins and nucleic acids.

[0053] As further disclosed below, the flocculation step may further include adjusting the pH before or after the addition of the flocculating agent. In particular, the solution may be acidified.

[0054] Additionally, the addition of flocculant and / or adjustment of pH can be carried out with the temperature adjusted to a desired level.

[0055] The following steps can be performed in any order: -addition of flocculant, then adjustment of pH, then adjustment of temperature or; -addition of flocculant, then adjustment of temperature, then adjustment of pH or; -adjusting the pH, then adding a flocculant, then adjusting the temperature, or; -adjusting the pH, then adjusting the temperature, then adding a flocculant or; -Adjusting the temperature, then adding a flocculant, then adjusting the pH, or; -Adjusting the temperature, then adjusting the pH, then adding the flocculant.

[0056] Additionally, after addition of flocculant and / or adjustment of pH, the solution may be held for some period of time to allow flocs to settle before downstream processing.

[0057] In the present invention, "flocculating agent" refers to an agent that can allow or promote flocculation in a solution containing a polysaccharide of interest together with contaminants by causing the aggregation of colloids and other suspended particles in the form of flocs or flakes, while allowing the polysaccharide of interest to remain in solution.

[0058] In one embodiment of the present invention, the flocculant comprises a polyvalent cation. In one embodiment, the flocculant is a polyvalent cation. In a preferred embodiment, the polyvalent 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 polyvalent cations selected from the group consisting of aluminum, iron, calcium, and magnesium. In one embodiment, the flocculant is a mixture of at least three polyvalent cations selected from the group consisting of aluminum, iron, calcium, and magnesium. In one embodiment, the flocculant is a mixture of four polyvalent cations selected from the group consisting of aluminum, iron, calcium, and magnesium.

[0059] In some embodiments, the flocculant comprises an agent selected from the group consisting of alum (e.g., potassium alum, sodium alum, or ammonium alum), aluminum chlorohydrate, aluminum sulfate, calcium oxide, calcium hydroxide, ferrous sulfate (iron sulfate), ferrous chloride (iron chloride), polyacrylamide, modified polyacrylamide, poly-DADMAC, polyethyleneimine (PEI), sodium aluminate, and sodium silicate. In some embodiments, the flocculant is selected from the group consisting of alum (e.g., potassium alum, sodium alum, or ammonium alum), aluminum chlorohydrate, aluminum sulfate, calcium oxide, calcium hydroxide, ferrous sulfate (iron sulfate), ferrous chloride (iron chloride), polyacrylamide, modified polyacrylamide, poly-DADMAC, sodium aluminate, and sodium silicate. In some embodiments, the flocculant is polyethyleneimine (PEI). In some embodiments, the flocculant comprises alum. In some embodiments, the flocculant is alum. In some embodiments, the flocculant includes potassium alum. In some embodiments, the flocculant is potassium alum. In some embodiments, the flocculant includes sodium alum. In some embodiments, the flocculant is sodium alum. In some embodiments, the flocculant includes ammonium alum. In some embodiments, the flocculant is ammonium alum.

[0060] In some embodiments, the flocculant is a mixture (e.g., two, three, or four agents) of agents selected from the group consisting of alum (e.g., potassium alum, sodium alum, or ammonium alum), aluminum chlorohydrate, aluminum sulfate, calcium oxide, calcium hydroxide, ferrous sulfate (II), ferrous chloride (III), polyacrylamide, modified polyacrylamide, polyDADMAC, polyethyleneimine (PEI), sodium aluminate, and sodium silicate. In some embodiments, the flocculant is selected from the group consisting of alum (e.g., potassium alum, sodium alum, or ammonium alum), aluminum chlorohydrate, aluminum sulfate, calcium oxide, calcium hydroxide, ferrous sulfate (II), ferrous chloride (III), polyacrylamide, modified polyacrylamide, polyDADMAC, sodium aluminate, and sodium silicate.

[0061] In some embodiments, 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 chlorohydrate, aluminum sulfate, calcium oxide, calcium hydroxide, ferrous sulfate (II) (iron sulfate), ferrous chloride (III) (iron chloride), polyacrylamide, modified polyacrylamide, polyDADMAC, sodium aluminate, and sodium silicate. In some embodiments, 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 chlorohydrate, aluminum sulfate, calcium oxide, calcium hydroxide, ferrous sulfate (II) (iron sulfate), ferrous chloride (III) (iron chloride), polyacrylamide, modified polyacrylamide, polyDADMAC, sodium aluminate, and sodium silicate.

[0062] In some embodiments, the flocculating agent comprises an agent selected from the group consisting of chitosan, isinglass, Moringa oleifera seed (Moringa oleifera), gelatin, Strychnos potatorum seed (Nirmali nut tree), guar gum, and alginates (e.g., wakame extract). In some embodiments, the flocculating agent comprises an agent selected from the group consisting of chitosan, isinglass, Moringa oleifera seed (Moringa oleifera), gelatin, Strychnos potatorum seed (Nirmali nut tree), guar gum, and alginates (e.g., wakame extract).

[0063] The concentration of the flocculating agent may depend on the agent used, the polysaccharide of interest, and the parameters of the flocculation step (eg, temperature).

[0064] In embodiments in which the flocculant includes or is alum, a flocculant concentration of about 0.1-20% (w / v) can be used. Preferably, a flocculant concentration of about 0.5-10% (w / v) is used. Even more preferably, a flocculant concentration of about 1-5% (w / v) is used. Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0065] In certain embodiments, the concentration is 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 Flocculant concentrations of 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) are used. In certain embodiments, the concentration is 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 16.0% (w / v), about 17.0% (w / v), about 18.0% (w / v), about 19.0% (w / v), about 20.0% (w / v), about 21.0% (w / v), about 22.0% (w / v), about 23.0% (w / v), about 24.0% (w / v), about 25.0% (w / v), about 26.0% ( Flocculant concentrations of 5.5% (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) are used. In some embodiments, flocculant concentrations 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) are used. In certain embodiments, 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.

[0066] In some embodiments of the present invention, the flocculant is added over a specified period of time. In some embodiments of the present invention, the flocculant is added over a period of a few seconds (e.g., 1-10 seconds) to about one month. In some embodiments of the present invention, the flocculant is added over a period of about 2 seconds to about two weeks. In some embodiments of the present invention, the flocculant is added over a period of about 1 minute to about one week. In some embodiments, the flocculant is added over a period of 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, The addition is carried out over a period of 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 to about 2 days.

[0067] Thus, in certain embodiments, the flocculant is added over a period of 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 to about 1 day.

[0068] Preferably, the flocculant is added over a period of 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 to about 1 day.

[0069] In certain embodiments, the flocculant is added over a period of about 15 minutes to about 3 hours, hi certain embodiments, the flocculant is added over a period of about 30 minutes to about 120 minutes.

[0070] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0071] In some embodiments, the flocculating agent may be added for 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, about 4 hours, about 4.5 hours, or 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.

[0072] In some embodiments, the flocculant is added without stirring. In other embodiments, the flocculant is added with stirring. In other embodiments, the flocculant is added with gentle stirring. In other embodiments, the flocculant is added with vigorous stirring.

[0073] The inventors have further surprisingly found that aggregation is improved when carried out at an acidic pH.

[0074] Thus, in embodiments of the invention, the aggregation step is carried out at a pH below 7.0, 6.0, 5.0, or 4.0. In certain embodiments of the invention, the aggregation step is carried out at a pH between 7.0 and 1.0. In certain embodiments, the aggregation step is carried out 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 certain embodiments, the aggregation step is carried out 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 some embodiments, the aggregation step is carried out at a pH of about 4.0, about 3.5, about 3.0, or about 2.5. In some embodiments, the aggregation step is carried out at a pH of about 3.5. Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0075] In some embodiments, the acidic pH is obtained by any of the methods in Section 1.1 above, or by acidifying a solution that has been further clarified as disclosed in Section 1.2 with an acid. In some embodiments, the acid is selected from the group consisting of HCl, H3PO4, citric acid, acetic acid, nitrous acid, and sulfuric acid. In some embodiments, the acid is an amino acid. In some embodiments, the acid is an amino acid selected from the group consisting of glycine, alanine, and glutamic acid. In some embodiments, the acid is HCl (hydrochloric acid). In some embodiments, the acid is sulfuric acid.

[0076] In some embodiments, the acid is added without stirring. Preferably, the acid is added with stirring. In some embodiments, the acid is added with gentle stirring. In some embodiments, the acid is added with vigorous stirring.

[0077] In some embodiments of the invention, after addition of flocculant (and optional acidification), the solution is held for some period of time to allow flocs to settle before downstream processing.

[0078] In some embodiments of the present invention, the aggregation step is carried out using a settling time of a few seconds (eg, 2-10 seconds) to about 1 minute. Preferably, the settling time is at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 65 minutes, at least about 70 minutes, at least about 75 minutes, at least about 80 minutes, at least about 85 minutes, at least about 90 minutes, at least about 95 minutes, at least about 100 minutes, at least about 105 minutes, at least about 110 minutes, at least about 115 minutes, at least about 120 minutes, at least about 125 minutes, at least about 130 minutes, at least about 135 minutes, at least about 140 minutes, at least about 145 minutes, at least about 150 minutes, at least about 155 minutes, or at least about 160 minutes. Preferably, the settling time is less than one week, although the settling time may be longer.

[0079] Thus, in certain embodiments, the settling time is about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 120 minutes, about 140 minutes, about 160 minutes, about 180 minutes, about 220 minutes, about 240 minutes, Approximately 300 minutes, approximately 360 minutes, approximately 420 minutes, approximately 480 minutes, approximately 540 minutes, approximately 600 minutes, approximately 660 minutes, approximately 720 minutes, approximately 780 minutes, approximately 840 minutes, approximately 900 minutes, approximately 960 minutes, approximately 1020 minutes, approximately 1080 minutes, approximately 1140 minutes, approximately 1200 minutes, approximately 1260 minutes, approximately 1320 minutes, approximately 1380 minutes, approximately 1440 minutes, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, or approximately 6 days to 1 week.

[0080] In some embodiments of the present invention, the settling time is from a few seconds (e.g., 1 to 10 seconds) to about 1 month. In some embodiments, the settling time is from about 2 seconds to about 2 weeks. In some embodiments of the present invention, the settling time is from about 1 minute to about 1 week. In some embodiments, the settling time is from 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, The time is 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 to about 2 days.

[0081] Thus, in certain embodiments, the settling time is from 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 to about 1 day.

[0082] Preferably, the sedimentation time is 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 to about 1 day.

[0083] In certain embodiments, the settling time is from about 15 minutes to about 3 hours, hi certain embodiments, the settling time is from about 30 minutes to about 120 minutes.

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

[0085] In certain embodiments, the settling 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 3 hours, about 3.5 hours, about 4 hours, or about 4. The preferred period is 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] Preferably, the settling time is about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, about 220 minutes, about 240 minutes, about 300 minutes, about 360 minutes, about 420 minutes, about 480 minutes, about 540 minutes, about 600 minutes, about 660 minutes, about 720 minutes, about 780 minutes, about 840 minutes, about 900 minutes, about 960 minutes, about 1020 minutes, about 1080 minutes, about 1140 minutes, about 1200 minutes, about 1260 minutes, about 1320 minutes, about 1380 minutes, or about 1440 minutes to 2 days. In certain embodiments, the settling time is about 5 minutes to about 1 day. In certain embodiments, the settling time is about 5 minutes to about 120 minutes.

[0087] The settling time may 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.

[0088] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0089] In some embodiments, the optional settling step is carried out without stirring. In some embodiments, the optional settling step is carried out with stirring. In other embodiments, the optional settling step is carried out with mild stirring. In other embodiments, the optional settling step is carried out with vigorous stirring.

[0090] In some embodiments of the present invention, the addition of the flocculant, settling of the solution, and / or adjustment of the pH is carried out at a temperature of about 4°C to about 30°C. In some embodiments, the addition of the flocculant, settling of the solution, and / or adjustment of the pH is carried out at 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 some embodiments, the addition of the flocculant, settling of the solution, and / or adjustment of the pH is carried out at a temperature of about 20°C. The inventors surprisingly discovered that flocculation can be further improved when carried out at elevated temperatures. Thus, in certain embodiments of the present invention, the addition of the flocculant, the settling of the solution, and / or the adjustment of the pH are carried out at a temperature of about 30° C. to about 95° C. In certain embodiments, the addition of the flocculant, the settling of the solution, and / or the adjustment of the pH are carried out at a temperature of about 35° C. to about 80° C., about 40° C. to about 70° C., about 45° C. to about 65° C., about 50° C. to about 60° C., about 50° C. to about 55° C., about 45° C. to about 55° C., or about 45° C. to about 55° C. In some embodiments, the addition of a flocculant, settling of the solution and / or adjustment of the pH is performed at temperatures of 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, In some embodiments, the addition of the flocculant, settling of the solution and / or adjustment of the pH is carried out at a temperature of about 50°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 some embodiments, the addition of the flocculant, settling of the solution and / or adjustment of the pH is carried out at a temperature of about 50°C.

[0091] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0092] In some embodiments, the addition of the flocculant is carried out at any of the temperatures described above.

[0093] In some embodiments, settling of the solution after addition of the flocculant is carried out at any of the temperatures described above.

[0094] In certain embodiments, the pH adjustment is carried out at any of the temperatures listed above.

[0095] In some embodiments, the addition of the flocculant and settling of the solution after addition of the flocculant is carried out at any of the temperatures described above.

[0096] In some embodiments, the addition of the flocculant and the adjustment of the pH are carried out at any of the temperatures listed above.

[0097] In some embodiments, the addition of the flocculant, settling of the solution after addition of the flocculant, and adjustment of the pH are carried out at any of the temperatures described above.

[0098] In some embodiments, the flocculation step involves adding a flocculating agent (as disclosed above) without adjusting the pH.

[0099] In some embodiments, the flocculation step involves adding a flocculant and allowing the solution to settle (as disclosed above) without adjusting the pH.

[0100] In some embodiments, the flocculation step includes adding a flocculant, adjusting the pH, and settling the solution (as disclosed above). In some embodiments, the flocculant is added before adjusting the pH. In other embodiments, the pH is adjusted before adding the flocculant.

[0101] In some embodiments, the flocculation step includes adding a flocculant, settling the solution, and adjusting the pH (as disclosed above). In some embodiments, adding the flocculant and settling the solution occurs before adjusting the pH. In other embodiments, the pH is adjusted before adding the flocculant and settling the solution. In some embodiments, adding the flocculant and adjusting the pH occurs before settling the solution. In other embodiments, the pH is adjusted before adding the flocculant and settling the solution.

[0102] In certain embodiments, the flocculation step includes adding a flocculating agent, adjusting the pH, and adjusting the temperature (as disclosed above).

[0103] These steps can be performed in any order: -addition of flocculant, then adjustment of pH, then adjustment of temperature or; -addition of flocculant, then adjustment of temperature, then adjustment of pH or; -adjusting the pH, then adding a flocculant, then adjusting the temperature, or; -adjusting the pH, then adjusting the temperature, then adding a flocculant or; -Adjusting the temperature, then adding a flocculant, then adjusting the pH, or; -Adjusting the temperature, then adjusting the pH, then adding the flocculant.

[0104] Additionally, after addition of flocculant and / or adjustment of pH, the solution may be held for some period of time to allow flocs to settle before downstream processing.

[0105] 1.3.Solid / liquid separation The agglomerated material can be separated from the polysaccharide of interest by any suitable solid / liquid separation method.

[0106] Thus, in an embodiment of the present invention, after flocculation, the suspension (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 further processing.

[0107] In an embodiment of the present invention, after flocculation, the suspension (obtained in section 1.2 above) is clarified by decantation. A decanter is used to separate the liquids when there is a sufficient density difference between them to allow the flocs to settle. In an operating decanter, three different zones will be present: a clear heavy liquid, a separating dispersed liquid (dispersion zone), and a clear light liquid. To produce a clear solution, it is generally necessary to leave a small amount of solution in the vessel. The decanter can be designed for continuous operation.

[0108] In an embodiment of the present invention, after flocculation, the suspension (obtained in section 1.2 above) is clarified by sedimentation (sedimentation). Sedimentation is the separation of suspended solid particles from a liquid mixture into a clear fluid and a slurry with a higher solids content by gravitational settling. Sedimentation can be carried out in a thickener, clarifier, or classifier. Because thickening and clarification are relatively inexpensive processes when used for the treatment of large volumes of liquid, they can be used for pre-concentration of feedstocks for filtration.

[0109] In embodiments of the invention, after flocculation, the suspension (obtained in section 1.2 above) is clarified by centrifugation. In some embodiments, the centrifugation is continuous centrifugation. In some embodiments, the centrifugation is bucket centrifugation. In some embodiments, the polysaccharide-containing supernatant is then collected for storage and / or further processing.

[0110] In some embodiments, the suspension is dissolved in 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 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.

[0111] In some embodiments, the suspension is centrifuged at about 5,000 g to about 25,000 g. In some embodiments, the suspension is centrifuged at about 8,000 g to about 20,000 g. In some embodiments, the suspension is centrifuged at about 10,000 g to about 15,000 g. In some embodiments, the suspension is centrifuged at about 10,000 g to about 12,000 g.

[0112] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0113] In some embodiments, the suspension is centrifuged for at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 65 minutes, at least 70 minutes, at least 75 minutes, at least 80 minutes, at least 85 minutes, at least 90 minutes, at least 95 minutes, at least 100 minutes, at least 105 minutes, at least 110 minutes, at least 115 minutes, at least 120 minutes, at least 125 minutes, at least 130 minutes, at least 135 minutes, at least 140 minutes, at least 145 minutes, at least 150 minutes, at least 155 minutes or at least 160 minutes.Preferably, the centrifugation time is less than 24 hours.

[0114] Thus, in certain embodiments, the suspension is centrifuged for 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 to 1440 minutes.

[0115] Preferably, the suspension is centrifuged for 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 to about 600 minutes. In certain embodiments, the suspension is centrifuged for about 5 minutes to about 3 hours. In certain embodiments, the suspension is centrifuged for about 5 minutes to about 120 minutes.

[0116] The suspension may be centrifuged for 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 to about 160 minutes.

[0117] The suspension may be centrifuged for 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 to about 60 minutes.

[0118] The suspension may be centrifuged for 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 minutes, about 1380 minutes or about 1440 minutes.

[0119] The suspension may be centrifuged for 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.

[0120] The suspension may be centrifuged for 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.

[0121] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0122] In an embodiment of the present invention, the centrifugation is continuous centrifugation. In the embodiment, the supply rate may be 50 to 5000 ml / min, 100 to 4000 ml / min, 150 to 3000 ml / min, 200 to 2500 ml / min, 250 to 2000 ml / min, 300 to 1500 ml / min, 300 to 1000 ml / min, 200 to 1000 ml / min, 200 to 1500 ml / min, 400 to 1500 ml / min, 500 to 1500 ml / min, 500 to 1000 ml / min, 500 to 2000 ml / min, 500 to 2500 ml / min, or 1000 to 2500 ml / min.

[0123] In certain embodiments, the feed rate is 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 1550, about 1600, about 1650, about 1700, about 1750, about 1800, about 1850, 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 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 5000, about 5500, about 6000, about 6500, about 7000, about 7500, about 8000, about 8500, about 90 The flow rate may be 0, 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.

[0124] In an embodiment of the present invention, after flocculation, the suspension (obtained in Section 1.2 above) is clarified by filtration. Filtration involves removing suspended solid particles in a liquid by passing the mixture through a porous medium that retains the particles and allows a clear filtrate to pass through. 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, which is dewatering filtration, or trapped within the filter medium, which is depth filtration. In one embodiment, after flocculation, the suspension (obtained in Section 1.2 above) is clarified by microfiltration. In one embodiment, the microfiltration is tangential flow microfiltration. In another embodiment, the microfiltration is dead-end filtration (vertical filtration). In one embodiment, the microfiltration is dead-end filtration that uses diatomaceous earth (DE), also known as DE diatomite, as a filter aid to facilitate and enhance the efficiency of solid / liquid separation. Thus, in one embodiment, after flocculation, the suspension (obtained in Section 1.2 above) is clarified by dead-end microfiltration with diatomaceous earth (DE). The DE can be impregnated (or contained) in a dead-end filter as an integral part of the depth filter.

[0125] Alternatively, DE can be added in powder form to the flocculation solution (obtained after section 1.2). In the latter case, the DE-treated flocculation solution can be further clarified by depth filtration.

[0126] In certain embodiments, the solution is processed by a microfiltration step in which the filter has a nominal retention range of about 0.01-2 μm, about 0.05-2 μm, about 0.1-2 μm, about 0.2-2 μm, about 0.3-2 μm, about 0.4-2 μm, about 0.45-2 μm, about 0.5-2 μm, about 0.6-2 μm, about 0.7-2 μm, about 0.8-2 μm, about 0.9-2 μm, about 1-2 μm, about 1.25-2 μm, about 1.5-2 μm, or about 1.75-2 μm.

[0127] In certain embodiments, the solution is processed by a microfiltration step in which the filter has a nominal retention range of about 0.01-1 μm, about 0.05-1 μm, about 0.1-1 μm, about 0.2-1 μm, about 0.3-1 μm, about 0.4-1 μm, about 0.45-1 μm, about 0.5-1 μm, about 0.6-1 μm, about 0.7-1 μm, about 0.8-1 μm, or about 0.9-1 μm.

[0128] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0129] In certain embodiments, the solution is processed by a microfiltration step in which the filter has a nominal retention range 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 μm.

[0130] In one embodiment, the solution is processed through a microfiltration step in which the filter has a nominal retention range of about 0.45 μm.

[0131] In one embodiment, the filter has a flow rate of 100 to 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 solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0132] In one embodiment, the filter has a flow rate of 100 to 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 to 2500 L / m 2 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0133] In one embodiment, the filter has a flow rate of 100 to 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 solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0134] In one embodiment, the filter has a flow rate of 100 to 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 solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0135] In one embodiment, the filter has a flow rate of 100 to 1000 L / m 2 , 200~1000L / m 2 , 300~1000L / m 2 , 400~1000L / m 2 , 500~1000L / m 2 or 750 to 1000 L / m 2 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0136] In one embodiment, the filter has a flow rate of 100 to 750 L / m 2 , 200~750L / m 2 , 300~750L / m 2 , 400~750L / m 2 or 500-750L / m 2 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0137] In one embodiment, the filter has a flow rate of 100 to 600 L / m 2 , 200~600L / m 2 , 300~600L / m 2 , 400~600L / m 2 or 400-600L / m 2 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0138] In one embodiment, the filter has a flow rate of 100 to 500 L / m 2 , 200~500L / m 2 , 300~500L / m 2 or 400-500L / m 2 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0139] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0140] In certain embodiments, the filter has a viscosity 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 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 / m2 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0141] The above solid / liquid separation methods can be used in standalone mode, or in combinations of two in any order, or in combinations of three in any order.

[0142] 1.4 Filtration (e.g., depth filtration) Once the solution has been processed through the flocculation step of Section 1.2 above and / or the solid / liquid separation step of Section 1.3 above, the polysaccharide-containing solution (eg, supernatant) can optionally be further clarified.

[0143] In some embodiments, the solution is filtered, thereby producing a further clarified solution. In some embodiments, filtration is applied directly to a solution obtained by any of the methods in Section 1.2 above. In some embodiments, filtration is applied to a solution that has been further clarified by the solid / liquid separation step described in Section 1.3 above.

[0144] In some embodiments, the solution is treated by a filtration step selected from the group consisting of depth filtration, filtration through activated carbon, size filtration, diafiltration, and ultrafiltration. In some embodiments, the solution is treated by a diafiltration step, particularly tangential flow filtration. In some embodiments, the solution is treated by a depth filtration step.

[0145] Depth filters use porous filtration media to retain particles throughout the media, rather than just on the surface of the media. Due to the complex and channel-like nature of the filtration media, particles are retained throughout the media within its structure, as opposed to on the surface.

[0146] In certain embodiments, the depth filter design processes the solution through a depth filtration step selected from the group consisting of a cassette, a cartridge, a deep bed (eg, a sand filter), and a lenticular filter.

[0147] In some embodiments, the depth filter has a diameter of about 0.01 to 100 μm, about 0.05 to 100 μm, about 0.1 to 100 μm, about 0.2 to 100 μm, about 0.3 to 100 μm, about 0.4 to 100 μm, about 0.5 to 100 μm, about 0.6 to 100 μm, about 0.7 to 100 μm, about 0.8 to 100 μm, about 0.9 to 100 μm, about 1 to 100 μm, about 1.25 to 100 μm, about 1.5 to 100 μm, about 1.75 to 100 μm, The solution is processed by a depth filtration step having a nominal retention range of about 2-100 μm, about 3-100 μm, about 4-100 μm, about 5-100 μm, about 6-100 μm, about 7-100 μm, about 8-100 μm, about 9-100 μm, about 10-100 μm, about 15-100 μm, about 20-100 μm, about 25-100 μm, about 30-100 μm, about 40-100 μm, about 50-100 μm or about 75-100 μm.

[0148] In some embodiments, the depth filter has a diameter of about 0.01 to 75 μm, about 0.05 to 75 μm, about 0.1 to 75 μm, about 0.2 to 75 μm, about 0.3 to 75 μm, about 0.4 to 75 μm, about 0.5 to 75 μm, about 0.6 to 75 μm, about 0.7 to 75 μm, about 0.8 to 75 μm, about 0.9 to 75 μm, about 1 to 75 μm, about 1.25 to 75 μm, about 1.5 to 75 μm, about 1.75 to 75 μm, about 1.85 to 75 μm, about 1.95 to 75 μm, about 2.05 to 2.55 μm, about 2.55 to 2.55 μm, about 2.65 to 2.75 μm, about 2.75 to 2.85 μm, about 2.85 to 2.95 μm, about 2.95 to 2.95 μm, about 3.05 to 3.0 ... The solution is processed by a depth filtration step having a nominal retention range of about 75 μm, about 2-75 μm, about 3-75 μm, about 4-75 μm, about 5-75 μm, about 6-75 μm, about 7-75 μm, about 8-75 μm, about 9-75 μm, about 10-75 μm, about 15-75 μm, about 20-75 μm, about 25-75 μm, about 30-75 μm, about 40-75 μm, or about 50-75 μm.

[0149] In some embodiments, the depth filter has a diameter of about 0.01 to 50 μm, about 0.05 to 50 μm, about 0.1 to 50 μm, about 0.2 to 50 μm, about 0.3 to 50 μm, about 0.4 to 50 μm, about 0.5 to 50 μm, about 0.6 to 50 μm, about 0.7 to 50 μm, about 0.8 to 50 μm, about 0.9 to 50 μm, about 1 to 50 μm, about 1.25 to 50 μm, about 1.5 to 50 μm, about 1.75 to 50 μm, about 1.85 to 50 μm, about 1.95 to 50 μm, about 2.05 to 50 μm, about 2.15 to 50 μm, about 2.25 to 50 μm, about 2.35 to 50 μm, about 2.45 to 50 μm, about 2.55 to 50 μm, about 2.65 to 50 μm, about 2.75 to 50 μm, about 2.85 to 50 μm, about 2.95 to 50 μm, about 3.05 to 50 μm, about 3.15 to 50 μm, about 3.25 to 50 μm, about 3.55 to 50 μm, about 3.65 to 50 μm, about 3.75 to 50 μm, about 3.85 to 50 μm, about 3.95 to 50 μm, about 3.95 to 50 μm, about 4.05 to 50 μm, about 4.05 to 50 μm, about 4.05 to 50 μm, about 4.05 The solution is processed by a depth filtration step with a nominal retention range of about 50 μm, about 2-50 μm, about 3-50 μm, about 4-50 μm, about 5-50 μm, about 6-50 μm, about 7-50 μm, about 8-50 μm, about 9-50 μm, about 10-50 μm, about 15-50 μm, about 20-50 μm, about 25-50 μm, about 30-50 μm, about 40-50 μm, about 50-50 μm.

[0150] In some embodiments, the depth filter has a diameter of about 0.01 to 25 μm, about 0.05 to 25 μm, about 0.1 to 25 μm, about 0.2 to 25 μm, about 0.3 to 25 μm, about 0.4 to 25 μm, about 0.5 to 25 μm, about 0.6 to 25 μm, about 0.7 to 25 μm, about 0.8 to 25 μm, about 0.9 to 25 μm, about 1 to 25 μm, or about 1.25 to 25 μm. The solution is processed by a depth filtration step having a nominal retention range of about 1.5-25 μm, about 1.75-25 μm, about 2-25 μm, about 3-25 μm, about 4-25 μm, about 5-25 μm, about 6-25 μm, about 7-25 μm, about 8-25 μm, about 9-25 μm, about 10-25 μm, about 15-25 μm, or about 20-25 μm.

[0151] In certain embodiments, the solution is processed by a depth filtration step, wherein the depth filter has a nominal retention range of about 0.01-10 μm, about 0.05-10 μm, about 0.1-10 μm, about 0.2-10 μm, about 0.3-10 μm, about 0.4-10 μm, about 0.5-10 μm, about 0.6-10 μm, about 0.7-10 μm, about 0.8-10 μm, about 0.9-10 μm, about 1-10 μm, about 1.25-10 μm, about 1.5-10 μm, about 1.75-10 μm, about 2-10 μm, about 3-10 μm, about 4-10 μm, about 5-10 μm, about 6-10 μm, about 7-10 μm, about 8-10 μm, or about 9-10 μm.

[0152] In certain embodiments, the solution is processed by a depth filtration step, wherein the depth filter has a nominal retention range of about 0.01-8 μm, about 0.05-8 μm, about 0.1-8 μm, about 0.2-8 μm, about 0.3-8 μm, about 0.4-8 μm, about 0.5-8 μm, about 0.6-8 μm, about 0.7-8 μm, about 0.8-8 μm, about 0.9-8 μm, about 1-8 μm, about 1.25-8 μm, about 1.5-8 μm, about 1.75-8 μm, about 2-8 μm, about 3-8 μm, about 4-8 μm, about 5-8 μm, about 6-8 μm, or about 7-8 μm.

[0153] In certain embodiments, the solution is processed by a depth filtration step in which the depth filter has a nominal retention range of about 0.01-5 μm, about 0.05-5 μm, about 0.1-5 μm, about 0.2-5 μm, about 0.3-5 μm, about 0.4-5 μm, about 0.5-5 μm, about 0.6-5 μm, about 0.7-5 μm, about 0.8-5 μm, about 0.9-5 μm, about 1-5 μm, about 1.25-5 μm, about 1.5-5 μm, about 1.75-5 μm, about 2-5 μm, about 3-5 μm, or about 4-5 μm.

[0154] In certain embodiments, the solution is processed by a depth filtration step in which the depth filter has a nominal retention range of about 0.01-2 μm, about 0.05-2 μm, about 0.1-2 μm, about 0.2-2 μm, about 0.3-2 μm, about 0.4-2 μm, about 0.5-2 μm, about 0.6-2 μm, about 0.7-2 μm, about 0.8-2 μm, about 0.9-2 μm, about 1-2 μm, about 1.25-2 μm, about 1.5-2 μm, about 1.75-2 μm, about 2-2 μm, about 3-2 μm, or about 4-2 μm.

[0155] In certain embodiments, the solution is processed by a depth filtration step in which the depth filter has a nominal retention range of about 0.01-1 μm, about 0.05-1 μm, about 0.1-1 μm, about 0.2-1 μm, about 0.3-1 μm, about 0.4-1 μm, about 0.5-1 μm, about 0.6-1 μm, about 0.7-1 μm, about 0.8-1 μm, or about 0.9-1 μm.

[0156] In certain embodiments, the solution is processed by a depth filtration step in which the depth filter has a nominal retention range of about 0.05-50 μm, 0.1-25 μm, 0.2-10 μm, 0.1-10 μm, 0.2-5 μm, or 0.25-1 μm.

[0157] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0158] In one embodiment, the depth filter has a flow rate of 1 to 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 The solution is processed by a depth filtration step with a filter capacity of 10 ...

[0159] In one embodiment, the depth filter has a flow rate of about 1 to 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 / m 2 , 200~1000L / m 2 , 300~1000L / m 2 , 400~1000L / m 2, 500~1000L / m 2 or 750 to 1000 L / m 2 The solution is processed by a depth filtration step with a filter capacity of 10 ...

[0160] In one embodiment, the depth filter has a flow capacity of about 1 to 750 L / m 2 , 5 to 750 L / 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 The solution is processed by a depth filtration step with a filter capacity of 10 ...

[0161] In one embodiment, the depth filter has a flow rate of 1 to 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 The solution is processed by a depth filtration step with a filter capacity of 10 ...

[0162] In one embodiment, the depth filter has a flow rate of 1 to 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 / m 2 , 150~400L / m 2 , 200~400L / m 2 or 300-400L / m 2 The solution is processed by a depth filtration step with a filter capacity of 10 ...

[0163] In one embodiment, the depth filter has a flow rate of 1 to 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 The solution is processed by a depth filtration step with a filter capacity of 10 ...

[0164] In one embodiment, the depth filter has a flow rate of 1 to 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 The solution is processed by a depth filtration step with a filter capacity of 10 ...

[0165] In one embodiment, the depth filter has a flow rate of 1 to 100 L / m 2 , 5 to 100 L / m 2 , 10~100L / m 2 , 25~100L / m 2 , 50~100L / m 2 or 75 to 100 L / m 2 The solution is processed by a depth filtration step with a filter capacity of 10 ...

[0166] In one embodiment, the depth filter has a flow rate of 1 to 50 L / m2 , 5~50L / m 2 , 10~50L / m 2 or 25 to 50 L / m 2 The solution is processed by a depth filtration step with a filter capacity of 10 ...

[0167] Any whole integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0168] In one embodiment, the feed rate is 1 to 1000 LMH (liters / m 2 / hr), 10-1000 LMH, 25-1000 LMH, 50-1000 LMH, 100-1000 LMH, 125-1000 LMH, 150-1000 LMH, 200-1000 LMH, 250-1000 LMH, 300-1000 LMH, 400-1000 LMH, 500-1000 LMH, 600-1000 LMH, 700-1000 LMH, 800-1000 LMH or 900-1000 LMH.

[0169] In some embodiments, the solution is treated by a depth filtration step with a feed rate of 1-500 LMH, 10-500 LMH, 25-500 LMH, 50-500 LMH, 100-500 LMH, 125-500 LMH, 150-500 LMH, 200-500 LMH, 250-500 LMH, 300-500 LMH, or 400-500 LMH.

[0170] In some embodiments, the solution is treated by a depth filtration step with a feed rate of 1-400 LMH, 10-400 LMH, 25-400 LMH, 50-400 LMH, 100-400 LMH, 125-400 LMH, 150-400 LMH, 200-400 LMH, 250-400 LMH, or 300-400 LMH.

[0171] In some embodiments, the solution is treated by a depth filtration step with a feed rate of 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.

[0172] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0173] In some embodiments, the feed rate may be 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, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 660, about 670, about 680, about 690, about 700, about 710, about 720, about 730, about 740, about 750, about 760, about 770, about 780, about 790, about 800, about 810, about 820, about 830, about 840, about 850 The solution is treated by a depth filtration step of about 10, 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.

[0174] 1.5 Optional Further Filtration Once the solution has been processed through the filtration step of Section 1.4 above, the resulting solution (ie, the filtrate) can optionally be further clarified.

[0175] In some embodiments, the solution is subjected to microfiltration. In some embodiments, the microfiltration is dead-end filtration (vertical filtration). In some embodiments, the microfiltration is tangential flow microfiltration.

[0176] In certain embodiments, the solution is processed by a microfiltration step in which the filter has a nominal retention range of about 0.01-2 μm, about 0.05-2 μm, about 0.1-2 μm, about 0.2-2 μm, about 0.3-2 μm, about 0.4-2 μm, about 0.45-2 μm, about 0.5-2 μm, about 0.6-2 μm, about 0.7-2 μm, about 0.8-2 μm, about 0.9-2 μm, about 1-2 μm, about 1.25-2 μm, about 1.5-2 μm, or about 1.75-2 μm.

[0177] In certain embodiments, the solution is processed by a depth filtration step in which the filter has a nominal retention range of about 0.01-1 μm, about 0.05-1 μm, about 0.1-1 μm, about 0.2-1 μm, about 0.3-1 μm, about 0.4-1 μm, about 0.45-1 μm, about 0.5-1 μm, about 0.6-1 μm, about 0.7-1 μm, about 0.8-1 μm, or about 0.9-1 μm.

[0178] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0179] In certain embodiments, the solution is processed by a microfiltration step in which the filter has a nominal retention range 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 μm.

[0180] In one embodiment, the solution is processed through a microfiltration step in which the filter has a nominal retention range of about 0.45 μm.

[0181] In one embodiment, the filter has a flow rate of 100 to 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 solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0182] In one embodiment, the filter has a flow rate of 100 to 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 to 2500 L / m 2 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0183] In one embodiment, the filter has a flow rate of 100 to 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 solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0184] In one embodiment, the filter has a flow rate of 100 to 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 solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0185] In one embodiment, the filter has a flow rate of 100 to 1000 L / m 2 , 200~1000L / m 2 , 300~1000L / m 2 , 400~1000L / m 2 , 500~1000L / m 2 or 750 to 1000 L / m 2 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0186] In one embodiment, the filter has a flow rate of 100 to 750 L / m 2 , 200~750L / m 2 , 300~750L / m 2 , 400~750L / m 2 or 500-750L / m 2 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0187] In one embodiment, the filter has a flow rate of 100 to 600 L / m 2 , 200~600L / m 2 , 300~600L / m 2 , 400~600L / m 2 or 400-600L / m 2 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0188] In one embodiment, the filter has a flow rate of 100 to 500 L / m 2 , 200~500L / m 2 , 300~500L / m 2 or 400-500L / m 2 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

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

[0190] In certain embodiments, the filter has a viscosity 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 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 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0191] 1.6 Ultrafiltration and / or diafiltration Once the solution has been filtered by any of the methods described in Section 1.4 above and / or by the filtration step described in Section 1.5 above, the resulting solution (i.e., filtrate) may optionally be further clarified by ultrafiltration and / or diafiltration.

[0192] Ultrafiltration (UF) is a process for concentrating dilute product streams. UF separates molecules in a solution based on the pore size or molecular weight cut-off (MWCO) of the membrane.

[0193] In an embodiment of the present invention, the solution (eg, the filtrate obtained in section 1.5 or 1.6 above) is treated by ultrafiltration.

[0194] In some embodiments, the solution is treated by ultrafiltration, and the membrane has a molecular weight cutoff in the range of about 5 kDa to 1000 kDa. In some embodiments, the membrane has a molecular weight cutoff in the range of about 10 kDa to 750 kDa. In some embodiments, the membrane has a molecular weight cutoff in the range of about 10 kDa to 500 kDa. In some embodiments, the membrane has a molecular weight cutoff in the range of about 10 kDa to 300 kDa. In some embodiments, the membrane has a molecular weight cutoff in the range of about 10 kDa to 100 kDa. In some embodiments, the membrane has a molecular weight cutoff in the range of about 10 kDa to 50 kDa. In some embodiments, the membrane has a molecular weight cutoff in the range of about 10 kDa to 30 kDa. In certain embodiments, the molecular weight cutoff of the membrane is in the range of about 5 kDa to 1000 kDa, about 10 kDa to 1000 kDa, about 20 kDa to 1000 kDa, about 30 kDa to 1000 kDa, about 40 kDa to 1000 kDa, about 50 kDa to 1000 kDa, about 75 kDa to 1000 kDa, about 100 kDa to 1000 kDa, about 150 kDa to 1000 kDa, about 200 kDa to 1000 kDa, about 300 kDa to 1000 kDa, about 400 kDa to 1000 kDa, about 500 kDa to 1000 kDa, or about 750 kDa to 1000 kDa.

[0195] In certain embodiments, the molecular weight cutoff of the membrane is in the range of about 5 kDa to 500 kDa, about 10 kDa to 500 kDa, about 20 kDa to 500 kDa, about 30 kDa to 500 kDa, about 40 kDa to 500 kDa, about 50 kDa to 500 kDa, about 75 kDa to 500 kDa, about 100 kDa to 500 kDa, about 150 kDa to 500 kDa, about 200 kDa to 500 kDa, about 300 kDa to 500 kDa, or about 400 kDa to 500 kDa.

[0196] In certain embodiments, the molecular weight cutoff of the membrane is in the range of about 5 kDa to 300 kDa, about 10 kDa to 300 kDa, about 20 kDa to 300 kDa, about 30 kDa to 300 kDa, about 40 kDa to 300 kDa, about 50 kDa to 300 kDa, about 75 kDa to 300 kDa, about 100 kDa to 300 kDa, about 150 kDa to 300 kDa, or about 200 kDa to 300 kDa.

[0197] In certain embodiments, the molecular weight cutoff of the membrane is in the range of about 5 kDa to 100 kDa, about 10 kDa to 100 kDa, about 20 kDa to 100 kDa, about 30 kDa to 100 kDa, about 40 kDa to 100 kDa, about 50 kDa to 100 kDa, or about 75 kDa to 100 kDa.

[0198] In certain embodiments, 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.

[0199] In some embodiments, the concentration factor of the ultrafiltration step is about 1.5 to 10. In some embodiments, the concentration factor is about 2 to 8. In some embodiments, the concentration factor is about 2 to 5.

[0200] In some embodiments, the enrichment 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 some embodiments, the enrichment factor is about 2, about 3, about 4, about 5, or about 6.

[0201] In an embodiment of the present invention, the solution (eg, the filtrate obtained in section 1.4 or 1.5 above) is processed by diafiltration.

[0202] In an embodiment of the present invention, the solution obtained after ultrafiltration (UF) as disclosed above in this section is further processed by diafiltration (UF / DF processing).

[0203] Diafiltration (DF) is used to exchange a product into a desired buffer solution (or water alone). In one embodiment, DF is used to change the chemical properties of a solution held at a constant volume. Undesired particles pass through the membrane, but the composition of the feed stream is changed to a more desirable state by the addition of a replacement fluid (buffer solution, salt solution, buffered salt solution, or water).

[0204] In some embodiments, the replacement fluid is water.

[0205] In some embodiments, the replacement fluid is salt water. In some embodiments, the salt is selected from the group consisting of magnesium chloride, potassium chloride, sodium chloride, and combinations thereof. In one particular embodiment, the salt is sodium chloride. In one embodiment, the replacement fluid 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 one particular 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.

[0206] In some embodiments, the replacement liquid is a buffer solution, and the buffering agent 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, amediol), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), N,N -Bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), sodium bicarbonate (bicarbonate), N,N'-bis(2-hydroxyethyl)-glycine (bicine), [bis-(2-hydroxyethyl)-imino]-tris-(hydroxymethylmethane) (bis-tris), 1,3-bis[tris(hydroxymethyl)-methylamino]propane (bis-tris-propane), boric acid, dimethylarsinic acid (cacodylate), 3-(cyclohexylamino)-propanesulfonic acid (CAPS), 3-(cyclohexylamino)-propanesulfonic acid N-(2-hydroxyethyl)-piperazine-N'-ethanesulfonic acid (HEPES), N-(2-hydroxyethyl)-piperazine-N'-3-propanesulfonic acid (HEPP), ... S, 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,The buffer solution is selected from the group consisting of 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 (tricine), and tris(hydroxymethyl)-aminomethane (tris).

[0207] In one embodiment, the diafiltration buffer is selected from the group consisting of a salt of acetic acid (acetate), a salt of citric acid (citrate), a salt of formic acid (formate), a salt of malic acid (malate), a salt of maleic acid (maleate), a salt of phosphoric acid (phosphate), and a salt of succinic acid (succinate). 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.

[0208] In certain embodiments, the pH of the diafiltration buffer is about 4.0 to 11.0, about 5.0 to 10.0, about 5.5 to 9.0, about 6.0 to 8.0, about 6.0 to 7.0, about 6.5 to 7.5, about 6.5 to 7.0, or about 6.0 to 7.5. Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0209] In some embodiments, 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 some embodiments, 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 some embodiments, the pH of the diafiltration buffer is about 6.5, about 7.0, or about 7.5. In some embodiments, the pH of the diafiltration buffer is about 7.0.

[0210] In certain embodiments, the concentration of the diafiltration buffer is about 0.01 mM to 100 mM, about 0.1 mM to 100 mM, about 0.5 mM to 100 mM, about 1 mM to 100 mM, about 2 mM to 100 mM, about 3 mM to 100 mM, about 4 mM to 100 mM, about 5 mM to 100 mM, about 6 mM to 100 mM, about 7 mM to 100 mM, about 8 mM to 100 mM, about 9 mM to 100 mM, about 10 mM to 100 mM, about 11 mM to 100 mM, about 12 mM to 100 mM, about 13 mM to 100 mM, about 14 mM to 100 mM, about 15 mM to 100 mM, about 16 mM to 100 mM, or about 17 mM to 100 mM. 100 mM, about 17 mM to 100 mM, about 18 mM to 100 mM, about 19 mM to 100 mM, about 20 mM to 100 mM, about 25 mM to 100 mM, about 30 mM to 100 mM, about 35 mM to 100 mM, about 40 mM to 100 mM, about 45 mM to 100 mM, about 50 mM to 100 mM, about 55 mM to 100 mM, about 60 mM to 100 mM, about 65 mM to 100 mM, about 70 mM to 100 mM, about 75 mM to 100 mM, about 80 mM to 100 mM, about 85 mM to 100 mM, about 90 mM to 100 mM, or about 95 mM to 100 mM.

[0211] In certain embodiments, the concentration of the diafiltration buffer is between about 0.01 mM and 50 mM, between about 0.1 mM and 50 mM, between about 0.5 mM and 50 mM, between about 1 mM and 50 mM, between about 2 mM and 50 mM, between about 3 mM and 50 mM, between about 4 mM and 50 mM, between about 5 mM and 50 mM, between about 6 mM and 50 mM, between about 7 mM and 50 mM, between about 8 mM and 50 mM, between about 9 mM and 50 mM, between about 10 mM and 50 mM, between about 11 mM and 50 mM, about 10 mM to 50 mM, about 12 mM to 50 mM, about 13 mM to 50 mM, about 14 mM to 50 mM, about 15 mM to 50 mM, about 16 mM to 50 mM, about 17 mM to 50 mM, about 18 mM to 50 mM, about 19 mM to 50 mM, about 20 mM to 50 mM, about 25 mM to 50 mM, about 30 mM to 50 mM, about 35 mM to 50 mM, about 40 mM to 50 mM, and about 45 mM to 50 mM.

[0212] In certain embodiments, the concentration of the diafiltration buffer is about 0.01 mM to 25 mM, about 0.1 mM to 25 mM, about 0.5 mM to 25 mM, about 1 mM to 25 mM, about 2 mM to 25 mM, about 3 mM to 25 mM, about 4 mM to 25 mM, about 5 mM to 25 mM, about 6 mM to 25 mM, about 7 mM to 25 mM, or about 8 mM to 25 mM. , about 9 mM to 25 mM, about 10 mM to 25 mM, about 11 mM to 25 mM, about 12 mM to 25 mM, about 13 mM to 25 mM, about 14 mM to 25 mM, about 15 mM to 25 mM, about 16 mM to 25 mM, about 17 mM to 25 mM, about 18 mM to 25 mM, about 19 mM to 25 mM, or about 20 mM to 25 mM.

[0213] In certain embodiments, the concentration of the diafiltration buffer is about 0.01 mM to 15 mM, about 0.1 mM to 15 mM, about 0.5 mM to 15 mM, about 1 mM to 15 mM, about 2 mM to 15 mM, about 3 mM to 15 mM, about 4 mM to 15 mM, about 5 mM to 15 mM, about 6 mM to 15 mM, about 7 mM to 15 mM, about 8 mM to 15 mM, about 9 mM to 15 mM, about 10 mM to 15 mM, about 11 mM to 15 mM, about 12 mM to 15 mM, about 13 mM to 15 mM, or about 14 mM to 15 mM.

[0214] In certain embodiments, the concentration of the diafiltration buffer is about 0.01 mM to 10 mM, about 0.1 mM to 10 mM, about 0.5 mM to 10 mM, about 1 mM to 10 mM, about 2 mM to 10 mM, about 3 mM to 10 mM, about 4 mM to 10 mM, about 5 mM to 10 mM, about 6 mM to 10 mM, about 7 mM to 10 mM, about 8 mM to 10 mM, or about 9 mM to 10 mM.

[0215] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0216] In certain embodiments, 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 , 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 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.

[0217] In certain embodiments, 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.

[0218] In one embodiment, the concentration of the diafiltration buffer is about 10 mM.

[0219] In some embodiments, the replacement liquid includes a chelating agent. In some embodiments, the replacement liquid includes an alum chelating agent. In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA), N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (EDTA-OH), hydroxyethylenediaminetriacetic acid (HEDTA), ethyleneglycol-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'-dipropionate. dihydrochloride (EDDP), ethylenediamine-tetrakis(methylenesulfonic acid) (EDTPO), nitrilotris(methylenephosphonic acid) (NTPO), imino-diacetic acid (IDA), hydroxyamino-diacetic acid (HIDA), nitrilo-triacetic acid (NTP), triethylenetetramine-hexaacetic acid (TTHA), dimercaptosuccinic acid (DMSA), 2,3-dimercapto-1-propanesulfonic acid (DMPS), alpha lipoic acid (ALA), nitrilotriacetic acid (NTA), thiamine tetrahydrofurfuryl disulfide (TTFD), dimercaprol, penicillamine, deferoxamine (DFOA), deferasirox, phosphonates, salts of citric acid (citrates), and combinations thereof.

[0220] 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 (citrates), and combinations thereof.

[0221] In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0222] In some embodiments, the chelating agent is a salt of citric acid (citrate), hi some embodiments, the chelating agent is sodium citrate.

[0223] Generally, the chelating agent is used at a concentration of 1 to 500 mM. In some embodiments, the concentration of the chelating agent in the replacement fluid is 2 to 400 mM. In some embodiments, the concentration of the chelating agent in the replacement fluid is 10 to 400 mM. In some embodiments, the concentration of the chelating agent in the replacement fluid is 10 to 200 mM. In some embodiments, the concentration of the chelating agent in the replacement fluid is 10 to 100 mM. In some embodiments, the concentration of the chelating agent in the replacement fluid is 10 to 50 mM. In some embodiments, the concentration of the chelating agent in the replacement fluid is 10 to 30 mM.

[0224] In certain embodiments, 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, or about 20 mM. M, 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 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.

[0225] In certain embodiments, 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.

[0226] In certain embodiments, the concentration of the chelating agent in the replacement fluid 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.

[0227] In some embodiments, 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 one particular embodiment, the salt is sodium chloride. In some embodiments, 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 one 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.

[0228] In embodiments of the invention, the number of dialysis volumes is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50. In embodiments of the invention, the number of dialysis volumes 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 embodiments of the invention, the number of dialysis volumes is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15.

[0229] In embodiments of the invention, the ultrafiltration and diafiltration steps are carried out at a temperature of about 20° C. to about 90° C. In certain embodiments, the ultrafiltration and diafiltration steps are carried out at a temperature of about 35° C. to about 80° C., about 40° C. to about 70° C., about 45° C. to about 65° C., about 50° C. to about 60° C., about 50° C. to about 55° C., about 45° C. to about 55° C., or about 45° C. to about 55° C.

[0230] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0231] In certain embodiments, the ultrafiltration and diafiltration steps are carried out at temperatures of 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, The ultrafiltration and diafiltration steps are carried out at a temperature of 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 certain embodiments, the ultrafiltration and diafiltration steps are carried out at a temperature of about 50° C.

[0232] In embodiments of the invention, the diafiltration step is carried out at a temperature of about 20° C. to about 90° C. In certain embodiments, the diafiltration step is carried out at a temperature of about 35° C. to about 80° C., about 40° C. to about 70° C., about 45° C. to about 65° C., about 50° C. to about 60° C., about 50° C. to about 55° C., about 45° C. to about 55° C., or about 45° C. to about 55° C.

[0233] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0234] In certain embodiments, the diafiltration step is carried out 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, about 80°C, about 81°C, about 82°C, about 83°C, about 84°C, about 85°C, about 86°C, about 87°C, about 88°C, about 89°C, about 90°C, about In certain embodiments, 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 certain embodiments, the diafiltration step is performed at a temperature of about 50°C.

[0235] In certain embodiments, the ultrafiltration step is carried out at a temperature of about 20° C. to about 90° C. In certain embodiments, the ultrafiltration step is carried out at a temperature of about 35° C. to about 80° C., about 40° C. to about 70° C., about 45° C. to about 65° C., about 50° C. to about 60° C., about 50° C. to about 55° C., about 45° C. to about 55° C., or about 45° C. to about 55° C. Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0236] In certain embodiments, the ultrafiltration step is carried out at temperatures of 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, about 80°C, about 81°C, about 82°C, about 83°C, about 84°C, about 85°C, about 86°C, about 87°C, about 88°C, about 89°C, about 90°C, In some embodiments, the ultrafiltration step is carried out 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 some embodiments, the ultrafiltration step is carried out at a temperature of about 50°C.

[0237] 1.7 Activated carbon filtration Once the solution has been treated by the flocculation step in section 1.2 above, the polysaccharide-containing solution can optionally be further clarified by an activated carbon filtration step.

[0238] In some embodiments, the solution (e.g., supernatant) from Section 1.2 that has been further processed by the solid / liquid separation step of Section 1.3 is further clarified by an activated charcoal filtration step. In some embodiments, the solution that has been further filtered by any of the methods described in Section 1.4 above and / or by the filtration step described in Section 1.5 above is further clarified by an activated charcoal filtration step. In some embodiments, the solution that has been further clarified by the ultrafiltration and / or diafiltration steps described in Section 1.6 above is further clarified by an activated charcoal filtration step.

[0239] 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 WO2008 / 118752).

[0240] In some embodiments, activated carbon (also called active charcoal) is added to the solution in an amount sufficient to adsorb the majority of protein and nucleic acid contaminants, and then removed once the contaminants have adsorbed onto the activated carbon. In some embodiments, the activated carbon is added in powder form, as a granular activated carbon layer, or as a compressed or extruded carbon block (see, e.g., Norit's active charcoal). In some embodiments, the activated carbon is added in an amount of about 0.1-20% (by weight), 1-15% (by weight), 1-10% (by weight), 2-10% (by weight), 3-10% (by weight), 4-10% (by weight), 5-10% (by weight), 1-5% (by weight), or 2-5% (by weight). The mixture is then stirred and allowed to settle. In some embodiments, the mixture is allowed to settle for about 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, 240 minutes, or longer. The activated carbon is then removed, which can be removed, for example, by centrifugation or filtration.

[0241] In a preferred embodiment, the solution is filtered through activated carbon immobilized in a matrix. The matrix may be any porous filter medium permeable to the solution. The matrix may include a support material and / or a binder material. The support material may be a synthetic polymer or a polymer of natural origin. Suitable synthetic polymers may include polystyrene, polyacrylamide, and polymethyl methacrylate, while naturally occurring polymers may include cellulose, polysaccharides, dextran, and agarose. Typically, the polymer support material is in the form of a fibrous network to provide mechanical rigidity. The binder material may be a resin. The matrix may be in the form of a membrane sheet. In one embodiment, the activated carbon immobilized in the matrix is ​​in the form of a flow-through carbon cartridge. The cartridge is a self-contained entity containing powdered activated carbon immobilized in the matrix and prepared in the form of a membrane sheet. The membrane sheet can be captured in a plastic permeable support to form a disk.

[0242] Alternatively, the membrane sheet may be spirally wound. To increase the filter surface area, several disks may be stacked on top of each other. In particular, the stacked disks have a central core pipe for collecting and removing the carbon-treated sample from the filter. The stacked disks may have a lenticular configuration.

[0243] The activated carbon in the carbon filter may be derived from a variety of raw materials, such as peat, lignite, wood, or coconut shells.

[0244] The charcoal can be activated using any process known in the art, such as steam or chemical treatment (eg, wood-based phosphate activated charcoal).

[0245] In the present invention, activated carbon immobilized in a matrix can be placed in a housing to form independent filter units. Each filter unit has its own inlet and outlet for the solution to be purified. Examples of filter units that can be used in the present invention are carbon cartridges from Cuno Inc. (Meriden, USA) or Pall Corporation (East Hill, USA). In particular, CUNO zetacarbon filters are suitable for use in the present invention. These carbon filters contain a cellulose matrix in which activated carbon powder is trapped in place and bound to a resin.

[0246] In one embodiment, the activated carbon filter disclosed above has a particle size of about 0.01 to 100 μm, about 0.05 to 100 μm, about 0.1 to 100 μm, about 0.2 to 100 μm, about 0.3 to 100 μm, about 0.4 to 100 μm, about 0.5 to 100 μm, about 0.6 to 100 μm, about 0.7 to 100 μm, about 0.8 to 100 μm, about 0.9 to 100 μm, about 1 to 100 μm, about 1.25 to 100 μm, about 1.5 to 100 μm, and having a nominal micron rating of about 1.75 to 100 μm, about 2 to 100 μm, about 3 to 100 μm, about 4 to 100 μm, about 5 to 100 μm, about 6 to 100 μm, about 7 to 100 μm, about 8 to 100 μm, about 9 to 100 μm, about 10 to 100 μm, about 15 to 100 μm, about 20 to 100 μm, about 25 to 100 μm, about 30 to 100 μm, about 40 to 100 μm, about 50 to 100 μm or about 75 to 100 μm.

[0247] In one embodiment, the activated carbon filter disclosed above has a particle size of about 0.01 to 50 μm, about 0.05 to 50 μm, about 0.1 to 50 μm, about 0.2 to 50 μm, about 0.3 to 50 μm, about 0.4 to 50 μm, about 0.5 to 50 μm, about 0.6 to 50 μm, about 0.7 to 50 μm, about 0.8 to 50 μm, about 0.9 to 50 μm, about 1 to 50 μm, about 1.25 to 50 μm, about 1.5 and has a nominal micron rating of about 1.75 to 50 μm, about 2 to 50 μm, about 3 to 50 μm, about 4 to 50 μm, about 5 to 50 μm, about 6 to 50 μm, about 7 to 50 μm, about 8 to 50 μm, about 9 to 50 μm, about 10 to 50 μm, about 15 to 50 μm, about 20 to 50 μm, about 25 to 50 μm, about 30 to 50 μm, about 40 to 50 μm or about 50 to 50 μm.

[0248] In one embodiment, the activated carbon filter disclosed above has a particle size of about 0.01 to 25 μm, about 0.05 to 25 μm, about 0.1 to 25 μm, about 0.2 to 25 μm, about 0.3 to 25 μm, about 0.4 to 25 μm, about 0.5 to 25 μm, about 0.6 to 25 μm, about 0.7 to 25 μm, about 0.8 to 25 μm, about 0.9 to 25 μm, about 1 to 25 μm, about 1 to 25 μm, about 2 ... μm, about 1.25 to 25 μm, about 1.5 to 25 μm, about 1.75 to 25 μm, about 2 to 25 μm, about 3 to 25 μm, about 4 to 25 μm, about 5 to 25 μm, about 6 to 25 μm, about 7 to 25 μm, about 8 to 25 μm, about 9 to 25 μm, about 10 to 25 μm, about 15 to 25 μm or about 20 to 25 μm.

[0249] In certain embodiments, the activated carbon filters disclosed above have a nominal micron rating of about 0.01-10 μm, about 0.05-10 μm, about 0.1-10 μm, about 0.2-10 μm, about 0.3-10 μm, about 0.4-10 μm, about 0.5-10 μm, about 0.6-10 μm, about 0.7-10 μm, about 0.8-10 μm, about 0.9-10 μm, about 1-10 μm, about 1.25-10 μm, about 1.5-10 μm, about 1.75-10 μm, about 2-10 μm, about 3-10 μm, about 4-10 μm, about 5-10 μm, about 6-10 μm, about 7-10 μm, about 8-10 μm, or about 9-10 μm.

[0250] In certain embodiments, the activated carbon filters disclosed above have a nominal micron rating of about 0.01-8 μm, about 0.05-8 μm, about 0.1-8 μm, about 0.2-8 μm, about 0.3-8 μm, about 0.4-8 μm, about 0.5-8 μm, about 0.6-8 μm, about 0.7-8 μm, about 0.8-8 μm, about 0.9-8 μm, about 1-8 μm, about 1.25-8 μm, about 1.5-8 μm, about 1.75-8 μm, about 2-8 μm, about 3-8 μm, about 4-8 μm, about 5-8 μm, about 6-8 μm, or about 7-8 μm.

[0251] In certain embodiments, the activated carbon filters disclosed above have a nominal micron rating of about 0.01-5 μm, about 0.05-5 μm, about 0.1-5 μm, about 0.2-5 μm, about 0.3-5 μm, about 0.4-5 μm, about 0.5-5 μm, about 0.6-5 μm, about 0.7-5 μm, about 0.8-5 μm, about 0.9-5 μm, about 1-5 μm, about 1.25-5 μm, about 1.5-5 μm, about 1.75-5 μm, about 2-5 μm, about 3-5 μm, or about 4-5 μm.

[0252] In certain embodiments, the activated carbon filters disclosed above have a nominal micron rating of about 0.01-2 μm, about 0.05-2 μm, about 0.1-2 μm, about 0.2-2 μm, about 0.3-2 μm, about 0.4-2 μm, about 0.5-2 μm, about 0.6-2 μm, about 0.7-2 μm, about 0.8-2 μm, about 0.9-2 μm, about 1-2 μm, about 1.25-2 μm, about 1.5-2 μm, about 1.75-2 μm, about 2-2 μm, about 3-2 μm, or about 4-2 μm.

[0253] In certain embodiments, the activated carbon filters disclosed above have a nominal micron rating of about 0.01 to 1 μm, about 0.05 to 1 μm, about 0.1 to 1 μm, about 0.2 to 1 μm, about 0.3 to 1 μm, about 0.4 to 1 μm, about 0.5 to 1 μm, about 0.6 to 1 μm, about 0.7 to 1 μm, about 0.8 to 1 μm, or about 0.9 to 1 μm.

[0254] In certain embodiments, the activated carbon filters disclosed above have a nominal micron rating of about 0.05-50 μm, 0.1-25 μm, 0.2-10 μm, 0.1-10 μm, 0.2-5 μm, or 0.25-1 μm.

[0255] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0256] In certain embodiments, the activated carbon filtration step is carried out at a feed rate of 1-500 LMH, 10-500 LMH, 15-500 LMH, 20-500 LMH, 25-500 LMH, 30-500 LMH, 40-500 LMH, 50-500 LMH, 100-500 LMH, 125-500 LMH, 150-500 LMH, 200-500 LMH, 250-500 LMH, 300-500 LMH, or 400-500 LMH.

[0257] In certain embodiments, the activated carbon filtration step is carried out at a feed rate of 1-200 LMH, 10-200 LMH, 15-200 LMH, 20-200 LMH, 25-200 LMH, 30-200 LMH, 40-200 LMH, 50-200 LMH, 100-200 LMH, 125-200 LMH, or 150-200 LMH.

[0258] In certain embodiments, the activated carbon filtration step is carried out at a feed rate of 1-150 LMH, 10-150 LMH, 15-150 LMH, 20-150 LMH, 25-150 LMH, 30-150 LMH, 40-150 LMH, 50-150 LMH, 100-150 LMH, or 125-150 LMH.

[0259] In some embodiments, the activated carbon filtration step is carried out at a feed rate of 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.

[0260] In certain embodiments, the activated carbon filtration step is carried out at a feed rate of 1-75 LMH, 5-75 LMH, 10-75 LMH, 15-75 LMH, 20-75 LMH, 25-75 LMH, 30-75 LMH, 35-75 LMH, 40-75 LMH, 45-75 LMH, 50-75 LMH, 55-75 LMH, 60-75 LMH, 65-75 LMH, or 70-75 LMH.

[0261] In certain embodiments, the activated carbon filtration step is carried out at a feed rate of 1-50 LMH, 5-50 LMH, 7-50 LMH, 10-50 LMH, 15-50 LMH, 20-50 LMH, 25-50 LMH, 30-50 LMH, 35-50 LMH, 40-50 LMH, or 45-50 LMH.

[0262] Any whole integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0263] In certain embodiments, the activated carbon filtration step is carried out 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.

[0264] In one embodiment, the filter has a flow rate of 5 to 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 to 100 L / m 2 The solution is treated with an activated carbon filter having a filtering capacity of 10 ...

[0265] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0266] In certain embodiments, 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 The solution is treated with an activated carbon filter having a filtering capacity of 10 ...

[0267] If the content of impurities is above a fixed threshold after the first activated carbon filtration step, the step may be repeated. In an embodiment of the present invention, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 activated carbon filtration steps are performed. In an embodiment of the present invention, 1, 2, or 3 activated carbon filtration steps are performed. In an embodiment of the present invention, 1 or 2 activated carbon filtration steps are performed.

[0268] In some embodiments, the solution is treated sequentially with activated carbon filters. In some embodiments, the solution is treated sequentially with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 activated carbon filters. In some embodiments, the solution is treated sequentially with 2, 3, 4, or 5 activated carbon filters. In some embodiments, the solution is treated sequentially with 2 activated carbon filters. In some embodiments, the solution is treated sequentially with 3 activated carbon filters. In some embodiments, the solution is treated sequentially with 4 activated carbon filters. In some embodiments, the solution is treated sequentially with 5 activated carbon filters.

[0269] In some embodiments, the activated carbon filtration step is performed in a single pass mode.

[0270] In another embodiment, the activated carbon filtration step is performed in recirculation mode. In the 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 cycles of activated carbon filtration are performed. In another embodiment, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles of activated carbon filtration are performed. In some embodiments, 2 or 3 cycles of activated carbon filtration are performed. In some embodiments, 2 cycles of activated carbon filtration are performed.

[0271] 1.8 Optional Further Filtering Once the solution has been treated with the activated carbon step in section 1.7 above, the resulting solution (ie, the filtrate) can optionally be further filtered.

[0272] In some embodiments, the solution is subjected to microfiltration, hi some embodiments, the microfiltration is dead-end filtration (vertical filtration).

[0273] In certain embodiments, the solution is processed by a microfiltration step in which the filter has a nominal retention range of about 0.01-2 μm, about 0.05-2 μm, about 0.1-2 μm, about 0.2-2 μm, about 0.3-2 μm, about 0.4-2 μm, about 0.45-2 μm, about 0.5-2 μm, about 0.6-2 μm, about 0.7-2 μm, about 0.8-2 μm, about 0.9-2 μm, about 1-2 μm, about 1.25-2 μm, about 1.5-2 μm, or about 1.75-2 μm.

[0274] In certain embodiments, the solution is processed by a microfiltration step in which the filter has a nominal retention range of about 0.01-1 μm, about 0.05-1 μm, about 0.1-1 μm, about 0.2-1 μm, about 0.3-1 μm, about 0.4-1 μm, about 0.45-1 μm, about 0.5-1 μm, about 0.6-1 μm, about 0.7-1 μm, about 0.8-1 μm, or about 0.9-1 μm.

[0275] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0276] In certain embodiments, the solution is processed by a microfiltration step in which 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 μm.

[0277] In one embodiment, the solution is processed through a microfiltration step in which the filter has a nominal retention rating of about 0.2 μm.

[0278] In one embodiment, the filter has a flow rate of 100 to 6000 L / m 2 , 200~6000L / m 2 , 300~6000L / m 2 , 400~6000L / m 2 , 500~6000L / m 2 , 750~6000L / m 2 , 1000~6000L / m 2 , 1500~6000L / m 2 , 2000~6000L / m 2 , 3000~6000L / m 2 or 4000~6000L / m 2 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0279] In one embodiment, the filter has a flow rate of 100 to 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 / m2 , 2500~4000L / m 2 , 3000~4000L / m 2 , 3000~4000L / m 2 or 3500-4000L / m 2 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0280] In one embodiment, the filter has a flow rate of 100 to 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 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0281] In one embodiment, the filter has a flow rate of 100 to 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 solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0282] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0283] In certain embodiments, the filter has a viscosity 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, about 2700, about 2800, 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 5100, about 5200, about 5300, about 5400, about 5500, about 5600, about 5700, about 5800, about 5900, about 6000, about 6100, about 6200, about 6300, about 6400, about 6500, about 6600, about 6700, about 6800, about 6900, about 7000, about 7100, about 7200, about 7300, about 7400 0, 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 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0284] 1.9 Hydrophobic Interaction Chromatography (HIC) This unit operation removes impurities with hydrophobic properties, such as residual lipopolysaccharides (endotoxins), remaining from previous purification steps.

[0285] Once the solution has been treated by the flocculation step in section 1.2 above, the polysaccharide-containing solution can optionally be further purified by an HIC step.

[0286] In some embodiments, the solution (e.g., supernatant) from Section 1.2 that has been further processed by the solid / liquid separation step in Section 1.3 is further purified by an HIC step. In some embodiments, a solution that has been further filtered by any of the methods described in Section 1.5 above and / or by the filtration step described in Section 1.4 above is further purified by an HIC step. In some embodiments, a solution that has been further clarified by the ultrafiltration and / or diafiltration steps described in Section 1.6 above is further purified by an HIC step. In some embodiments, a solution that has been further clarified by the activated charcoal filtration step in Section 1.7 is further purified by an HIC step.

[0287] In certain embodiments, the solution further clarified by the ultrafiltration and / or diafiltration steps described above in Section 1.6 can be further purified by an ion exchange membrane (IEX) filtration step followed by a HIC step.

[0288] In certain embodiments, the HIC step is performed using a hydrophobic adsorbent selected from the group consisting of, but not limited to, phenyl membrane, butyl-, phenyl-, and octyl-agarose, butyl-, phenyl-, ether-, polypropylene glycol-, and hexyl-organic polymer resins.

[0289] In certain embodiments, the hydrophobic adsorbent used in the HIC step is a phenyl membrane, such as a SARTOBIND Phenyl membrane or a CYTIVA Phenyl Adsorber membrane.

[0290] In one embodiment, the hydrophobic adsorbent used in the HIC step is a SARTOBIND Phenyl membrane.

[0291] In some embodiments, the material from the previous step (e.g., carbon filtrate) is treated with an equilibration buffer to obtain a running buffer containing the material to be purified and a desired salt concentration. In some embodiments, the equilibration buffer contains salt, and the final salt concentration (i.e., in the running buffer) is 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, or 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 running buffer has a pH of about 4.0 to about 8.0. In one embodiment, the pH of the running 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 certain embodiments, the equilibration buffer contains a salt selected from ammonium sulfate (preferably at a final concentration of 0.5 M to 3.0 M in the running buffer and at a pH of 6.0±2.0), sodium phosphate (preferably at a final concentration of 0.5 M to 3.0 M in the running buffer and at a pH of 7.0±1.5), potassium phosphate (preferably at a final concentration of 0.5 M to 3.0 M in the running buffer and at a pH of 7.0±1.5), sodium sulfate (preferably at a final concentration of 0.1 M to 0.75 M in the running buffer and at a pH of 6.0±2.0), sodium citrate (preferably at a final concentration of 0.1 M to 1.5 M in the running buffer and at a pH of 6.0±2.0), or sodium chloride (preferably at a final concentration of 0.5 M to 5.0 M in the running buffer and at a pH of 7.0±1.5).

[0292] In one embodiment, the equilibration buffer contains ammonium sulfate, and the final salt concentration in the running buffer is about 1.0 M to 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, or about 2.0 M.

[0293] In certain embodiments, the hydrophobic adsorbent is equilibrated with a running buffer, and then the material to be purified in the running buffer is passed through the column or membrane.

[0294] In one embodiment, the hydrophobic adsorbent is a phenyl membrane and the flow rate is about 0.1 to about 20 membrane volumes / min, about 0.1 to about 10 membrane volumes / min, about 0.2 to about 10 membrane volumes / min, about 0.2 to about 5 membrane volumes / min, or about 0.1 to about 1 membrane volume / min. In one embodiment, the hydrophobic adsorbent is a phenyl membrane and the flow rate is about 0.1 to about 1.0 membrane volumes / min, 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 volumes / min.

[0295] In some embodiments, the HIC membrane is then rinsed with running buffer and optionally further washed with water. The flow-through effluent along with the buffer rinse was collected as the HIC filtrate, and the water wash was also collected for analysis.

[0296] 1.10 Ultrafiltration / Diafiltration Once the solution has been treated by the HIC step described above in Section 1.9 and / or by the further filtration step described above in Section 1.8, the resulting solution (i.e., filtrate) may optionally be further clarified by ultrafiltration and / or diafiltration.

[0297] In an embodiment of the present invention, the solution (eg, obtained in section 1.9 or 1.8 above) is processed by ultrafiltration.

[0298] In some embodiments, the solution is treated by ultrafiltration, and the membrane has a molecular weight cutoff in the range of about 5 kDa to 1000 kDa. In some embodiments, the membrane has a molecular weight cutoff in the range of about 10 kDa to 750 kDa. In some embodiments, the membrane has a molecular weight cutoff in the range of about 10 kDa to 500 kDa. In some embodiments, the membrane has a molecular weight cutoff in the range of about 10 kDa to 300 kDa. In some embodiments, the membrane has a molecular weight cutoff in the range of about 10 kDa to 100 kDa. In some embodiments, the membrane has a molecular weight cutoff in the range of about 10 kDa to 50 kDa. In some embodiments, the membrane has a molecular weight cutoff in the range of about 10 kDa to 30 kDa. In certain embodiments, the molecular weight cutoff of the membrane is in the range of about 5 kDa to 1000 kDa, about 10 kDa to 1000 kDa, about 20 kDa to 1000 kDa, about 30 kDa to 1000 kDa, about 40 kDa to 1000 kDa, about 50 kDa to 1000 kDa, about 75 kDa to 1000 kDa, about 100 kDa to 1000 kDa, about 150 kDa to 1000 kDa, about 200 kDa to 1000 kDa, about 300 kDa to 1000 kDa, about 400 kDa to 1000 kDa, about 500 kDa to 1000 kDa, or about 750 kDa to 1000 kDa.

[0299] In certain embodiments, the molecular weight cutoff of the membrane is in the range of about 5 kDa to 500 kDa, about 10 kDa to 500 kDa, about 20 kDa to 500 kDa, about 30 kDa to 500 kDa, about 40 kDa to 500 kDa, about 50 kDa to 500 kDa, about 75 kDa to 500 kDa, about 100 kDa to 500 kDa, about 150 kDa to 500 kDa, about 200 kDa to 500 kDa, about 300 kDa to 500 kDa, or about 400 kDa to 500 kDa.

[0300] In certain embodiments, the molecular weight cutoff of the membrane is in the range of about 5 kDa to 300 kDa, about 10 kDa to 300 kDa, about 20 kDa to 300 kDa, about 30 kDa to 300 kDa, about 40 kDa to 300 kDa, about 50 kDa to 300 kDa, about 75 kDa to 300 kDa, about 100 kDa to 300 kDa, about 150 kDa to 300 kDa, or about 200 kDa to 300 kDa.

[0301] In certain embodiments, the molecular weight cutoff of the membrane is in the range of about 5 kDa to 100 kDa, about 10 kDa to 100 kDa, about 20 kDa to 100 kDa, about 30 kDa to 100 kDa, about 40 kDa to 100 kDa, about 50 kDa to 100 kDa, or about 75 kDa to 100 kDa.

[0302] In certain embodiments, 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.

[0303] In some embodiments, the concentration factor for the ultrafiltration step is about 1.5 to about 10.0. In some embodiments, the concentration factor is about 2.0 to about 8.0. In some embodiments, the concentration factor is about 2.0 to about 5.0.

[0304] In some embodiments, the enrichment 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 some embodiments, the enrichment factor is about 2.0, about 3.0, about 4.0, about 5.0, or about 6.0.

[0305] In an embodiment of the present invention, the solution (eg, the filtrate obtained in section 1.9 or 1.8 above) is processed by diafiltration.

[0306] In an embodiment of the present invention, the solution obtained after ultrafiltration (UF) as disclosed above in this section is further processed by diafiltration (UF / DF processing).

[0307] Diafiltration (DF) is used to exchange a product into a desired buffer solution (or water alone). In one embodiment, DF is used to change the chemical properties of a solution held at a constant volume. Undesired particles pass through the membrane, but the composition of the feed stream is changed to a more desirable state by the addition of a replacement fluid (buffer solution, salt solution, buffered salt solution, or water).

[0308] In some embodiments, the replacement fluid is water.

[0309] In one embodiment, the replacement fluid is salt water. In some embodiments, the salt is selected from the group consisting of magnesium chloride, potassium chloride, sodium chloride, and combinations thereof. In one particular embodiment, the salt is sodium chloride. In one embodiment, the replacement fluid 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 one particular embodiment, the replacement fluid 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 one particular embodiment, the replacement fluid 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.

[0310] In some embodiments, the replacement liquid is a buffer solution, and the buffering agent 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, amediol), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), N,N -Bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), sodium bicarbonate (bicarbonate), N,N'-bis(2-hydroxyethyl)-glycine (bicine), [bis-(2-hydroxyethyl)-imino]-tris-(hydroxymethylmethane) (bis-tris), 1,3-bis[tris(hydroxymethyl)-methylamino]propane (bis-tris-propane), boric acid, dimethylarsinic acid (cacodylate), 3-(cyclohexylamino)-propanesulfonic acid (CAPS), 3-(cyclohexylamino)-propanesulfonic acid N-(2-hydroxyethyl)-piperazine-N'-ethanesulfonic acid (HEPES), N-(2-hydroxyethyl)-piperazine-N'-3-propanesulfonic acid (HEPP), ... S, 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,The buffer solution is selected from the group consisting of 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 (tricine), and tris(hydroxymethyl)-aminomethane (tris).

[0311] In some embodiments, the diafiltration buffer is selected from the group consisting of a salt of acetic acid (acetate), a salt of citric acid (citrate), a salt of formic acid (formate), a salt of malic acid (malate), a salt of maleic acid (maleate), a salt of phosphoric acid (phosphate), and a salt of succinic acid (succinate). In some embodiments, the diafiltration buffer is a salt of citric acid (citrate). In some embodiments, the diafiltration buffer is a salt of succinic acid (succinate). In some embodiments, the salt is a sodium salt. In some embodiments, the salt is a potassium salt.

[0312] In certain embodiments, the pH of the diafiltration buffer is about 4.0 to 11.0, about 5.0 to 10.0, about 5.5 to 9.0, about 6.0 to 8.0, about 6.0 to 7.0, about 6.5 to 7.5, about 6.5 to 7.0, or about 6.0 to 7.5. Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0313] In some embodiments, 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 some embodiments, 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 some embodiments, the pH of the diafiltration buffer is about 6.5, about 7.0, or about 7.5. In some embodiments, the pH of the diafiltration buffer is about 6.0. In some embodiments, the pH of the diafiltration buffer is about 6.5. In some embodiments, the pH of the diafiltration buffer is about 7.0.

[0314] In certain embodiments, the concentration of the diafiltration buffer is about 0.01 mM to 100 mM, about 0.1 mM to 100 mM, about 0.5 mM to 100 mM, about 1 mM to 100 mM, about 2 mM to 100 mM, about 3 mM to 100 mM, about 4 mM to 100 mM, about 5 mM to 100 mM, about 6 mM to 100 mM, about 7 mM to 100 mM, about 8 mM to 100 mM, about 9 mM to 100 mM, about 10 mM to 100 mM, about 11 mM to 100 mM, about 12 mM to 100 mM, about 13 mM to 100 mM, about 14 mM to 100 mM, about 15 mM to 100 mM, about 16 mM to 100 mM, or about 17 mM to 100 mM. 100 mM, about 17 mM to 100 mM, about 18 mM to 100 mM, about 19 mM to 100 mM, about 20 mM to 100 mM, about 25 mM to 100 mM, about 30 mM to 100 mM, about 35 mM to 100 mM, about 40 mM to 100 mM, about 45 mM to 100 mM, about 50 mM to 100 mM, about 55 mM to 100 mM, about 60 mM to 100 mM, about 65 mM to 100 mM, about 70 mM to 100 mM, about 75 mM to 100 mM, about 80 mM to 100 mM, about 85 mM to 100 mM, about 90 mM to 100 mM, or about 95 mM to 100 mM.

[0315] In certain embodiments, the concentration of the diafiltration buffer is about 0.01 mM to 50 mM, about 0.1 mM to 50 mM, about 0.5 mM to 50 mM, about 1 mM to 50 mM, about 2 mM to 50 mM, about 3 mM to 50 mM, about 4 mM to 50 mM, about 5 mM to 50 mM, about 6 mM to 50 mM, about 7 mM to 50 mM, about 8 mM to 50 mM, about 9 mM to 50 mM, about 10 mM to 50 mM, or about 11 mM. about 12 mM to 50 mM, about 13 mM to 50 mM, about 14 mM to 50 mM, about 15 mM to 50 mM, about 16 mM to 50 mM, about 17 mM to 50 mM, about 18 mM to 50 mM, about 19 mM to 50 mM, about 20 mM to 50 mM, about 25 mM to 50 mM, about 30 mM to 50 mM, about 35 mM to 50 mM, about 40 mM to 50 mM, or about 45 mM to 50 mM.

[0316] In certain embodiments, the concentration of the diafiltration buffer is about 0.01 mM to 25 mM, about 0.1 mM to 25 mM, about 0.5 mM to 25 mM, about 1 mM to 25 mM, about 2 mM to 25 mM, about 3 mM to 25 mM, about 4 mM to 25 mM, about 5 mM to 25 mM, about 6 mM to 25 mM, about 7 mM to 25 mM, or about 8 mM to 25 mM. , about 9 mM to 25 mM, about 10 mM to 25 mM, about 11 mM to 25 mM, about 12 mM to 25 mM, about 13 mM to 25 mM, about 14 mM to 25 mM, about 15 mM to 25 mM, about 16 mM to 25 mM, about 17 mM to 25 mM, about 18 mM to 25 mM, about 19 mM to 25 mM, or about 20 mM to 25 mM.

[0317] In certain embodiments, the concentration of the diafiltration buffer is about 0.01 mM to 15 mM, about 0.1 mM to 15 mM, about 0.5 mM to 15 mM, about 1 mM to 15 mM, about 2 mM to 15 mM, about 3 mM to 15 mM, about 4 mM to 15 mM, about 5 mM to 15 mM, about 6 mM to 15 mM, about 7 mM to 15 mM, about 8 mM to 15 mM, about 9 mM to 15 mM, about 10 mM to 15 mM, about 11 mM to 15 mM, about 12 mM to 15 mM, about 13 mM to 15 mM, or about 14 mM to 15 mM.

[0318] In certain embodiments, the concentration of the diafiltration buffer is about 0.01 mM to 10 mM, about 0.1 mM to 10 mM, about 0.5 mM to 10 mM, about 1 mM to 10 mM, about 2 mM to 10 mM, about 3 mM to 10 mM, about 4 mM to 10 mM, about 5 mM to 10 mM, about 6 mM to 10 mM, about 7 mM to 10 mM, about 8 mM to 10 mM, or about 9 mM to 10 mM.

[0319] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0320] In certain embodiments, 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 , 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 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.

[0321] In some embodiments, the diafiltration buffer concentration 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 some embodiments, the diafiltration buffer concentration is about 30 mM. In some embodiments, the diafiltration buffer concentration is about 25 mM. In some embodiments, the diafiltration buffer concentration is about 20 mM. In some embodiments, the diafiltration buffer concentration is about 15 mM. In some embodiments, the diafiltration buffer concentration is about 10 mM.

[0322] In some embodiments, the diafiltration buffer solution contains a salt. In some embodiments, the salt is selected from the group consisting of magnesium chloride, potassium chloride, sodium chloride, and combinations thereof. In one particular embodiment, the salt is sodium chloride. In one particular embodiment, the diafiltration buffer solution contains 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.

[0323] In embodiments of the invention, the number of dialysis volumes is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50. In embodiments of the invention, the number of dialysis volumes 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 embodiments of the invention, the number of dialysis volumes is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15.

[0324] In embodiments of the invention, the ultrafiltration and diafiltration steps are carried out at a temperature of about 20° C. to about 90° C. In certain embodiments, the ultrafiltration and diafiltration steps are carried out at a temperature of about 35° C. to about 80° C., about 40° C. to about 70° C., about 45° C. to about 65° C., about 50° C. to about 60° C., about 50° C. to about 55° C., about 45° C. to about 55° C., or about 45° C. to about 55° C.

[0325] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0326] In certain embodiments, the ultrafiltration and diafiltration steps are carried out at temperatures of 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, The ultrafiltration and diafiltration steps are carried out at a temperature of 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 certain embodiments, the ultrafiltration and diafiltration steps are carried out at a temperature of about 50° C.

[0327] In embodiments of the invention, the diafiltration step is carried out at a temperature of about 20° C. to about 90° C. In certain embodiments, the diafiltration step is carried out at a temperature of about 35° C. to about 80° C., about 40° C. to about 70° C., about 45° C. to about 65° C., about 50° C. to about 60° C., about 50° C. to about 55° C., about 45° C. to about 55° C., or about 45° C. to about 55° C.

[0328] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0329] In certain embodiments, the diafiltration step is carried out 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, about 80°C, about 81°C, about 82°C, about 83°C, about 84°C, about 85°C, about 86°C, about 87°C, about 88°C, about 89°C, about 90°C, about In certain embodiments, 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 certain embodiments, the diafiltration step is performed at a temperature of about 50°C.

[0330] In certain embodiments, the ultrafiltration step is carried out at a temperature of about 20° C. to about 90° C. In certain embodiments, the ultrafiltration step is carried out at a temperature of about 35° C. to about 80° C., about 40° C. to about 70° C., about 45° C. to about 65° C., about 50° C. to about 60° C., about 50° C. to about 55° C., about 45° C. to about 55° C., or about 45° C. to about 55° C. Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0331] In certain embodiments, the ultrafiltration step is carried out at temperatures of 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, about 80°C, about 81°C, about 82°C, about 83°C, about 84°C, about 85°C, about 86°C, about 87°C, about 88°C, about 89°C, about 90°C, In some embodiments, the ultrafiltration step is carried out 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 some embodiments, the ultrafiltration step is carried out at a temperature of about 50°C.

[0332] 1.11 Homogenization / Sizing The polysaccharide may become slightly reduced in size during the purification procedure.

[0333] In one embodiment, the purified polysaccharide solution of the present invention (eg, obtained by ultrafiltration and / or diafiltration as described in Section 1.10) is not sized.

[0334] In some embodiments, the polysaccharides can be homogenized by a sizing technique. Mechanical or chemical sizing can be used. Chemical hydrolysis can be performed, for example, using acetic acid. Mechanical sizing can be performed using high pressure homogenization shear.

[0335] Thus, in one embodiment, the purified polysaccharide solution obtained by ultrafiltration and / or diafiltration as described in Section 1.10 is sized to a target molecular weight.

[0336] 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).

[0337] In some embodiments, the purified polysaccharide is sized to a molecular weight of about 5 kDa to about 4,000 kDa. In other such embodiments, the purified polysaccharide is sized to a molecular weight of about 10 kDa to about 4,000 kDa. In other such embodiments, the purified polysaccharide is sized to a molecular weight of about 50 kDa to about 4,000 kDa. In further such embodiments, the purified polysaccharide is sized to a molecular weight of about 50 kDa to about 3,500 kDa; about 50 kDa to about 3,000 kDa; about 50 kDa to about 2,500 kDa; about 50 kDa to about 2,000 kDa; about 50 kDa to about 1,750 kDa; about 50 kDa to about 1,500 kDa; about 50 kDa to about 1,250 kDa; or about 50 kDa to about 1,000 kDa. kDa; about 50kDa to about 750kDa; about 50kDa to about 500kDa; about 100kDa to about 4,000kDa; about 100kDa to about 3,500kDa; about 100kDa to about 3,000kDa; about 100kDa to about 2,500kDa; about 100kDa to about 2,250kDa; about 100kDa to about 2,000kDa; about 100kDa to about 1,750kDa a; about 100kDa to about 1,500kDa; about 100kDa to about 1,250kDa; about 100kDa to about 1,000kDa; about 100kDa to about 750kDa; about 100kDa ~about 500kDa; about 200kDa to about 4,000kDa; about 200kDa to about 3,500kDa; about 200kDa to about 3,000kDa; about 200kDa to about 2,500kDa a; sized to a molecular weight of about 200 kDa to about 2,250 kDa; about 200 kDa to about 2,000 kDa; about 200 kDa to about 1,750 kDa; about 200 kDa to about 1,500 kDa; about 200 kDa to about 1,250 kDa; about 200 kDa to about 1,000 kDa; about 200 kDa to about 750 kDa; or about 200 kDa to about 500 kDa.In further such embodiments, the purified polysaccharide has a molecular weight of from about 250 kDa to about 3,500 kDa; from about 250 kDa to about 3,000 kDa; from about 250 kDa to about 2,500 kDa; from about 250 kDa to about 2,000 kDa; from about 250 kDa to about 1,750 kDa; from about 250 kDa to about 1,500 kDa; from about 250 kDa to about 1,250 kDa; from about 250 kDa to about 1,000kDa; about 250kDa to about 750kDa; about 250kDa to about 500kDa; about 300kDa to about 4,000kDa; about 300kDa to about 3,500kDa; about 30 0kDa~about 3,000kDa;about 300kDa~about 2,500kDa;about 300kDa~about 2,250kDa;about 300kDa~about 2,000kDa;about 300kDa~about 1, 750kDa; approximately 300kDa to approximately 1,500kDa; approximately 300kDa to approximately 1,250kDa; approximately 300kDa to approximately 1,000kDa; approximately 300kDa to approximately 750kDa; 0kDa~about 500kDa;about 500kDa~about 4,000kDa;about 500kDa~about 3,500kDa;about 500kDa~about 3,000kDa;about 500kDa~about 2,50 about 500 kDa to about 2,250 kDa; about 500 kDa to about 2,000 kDa; about 500 kDa to about 1,750 kDa; about 500 kDa to about 1,500 kDa; about 500 kDa to about 1,250 kDa; about 500 kDa to about 1,000 kDa; about 500 kDa to about 750 kDa; or about 500 kDa to about 600 kDa.

[0338] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0339] In some embodiments, the purified polysaccharide is 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 700 kDa, about 750 kDa, about 800 kDa, about 850 kDa, about 900 kDa, about 950 kDa, about 1000 kDa, about 1250 kDa, about 1500 kDa, about 1750 kDa, about 2000 kDa, about 2250 kDa, about 2500 kDa, about 2750 kDa, about 3000 kDa, about 3250 kDa, about 3500 kDa, about 3750 kDa or about 4,000 kDa.

[0340] In a preferred embodiment, the purified polysaccharide is a capsular polysaccharide derived 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 capsular polysaccharide has a molecular weight within one of the ranges above or has a size approximately equal to the above ranges.

[0341] 1.12 Sterile filtration In one embodiment, the solution of the purified polysaccharide of the present invention is sterile filtered.

[0342] Thus, in one embodiment, ultrafiltration and / or diafiltration as described in Section 1.10 may optionally be followed by a sterile filtration step.

[0343] In one embodiment, if performed, the homogenization / sizing step described in Section 1.11 may be optionally followed by a sterile filtration step.

[0344] In one embodiment, any of the steps described in sections 1.2-1.9 may optionally be followed by a sterile filtration step.

[0345] In some embodiments, the sterile filtration is dead-end filtration (vertical filtration). In some embodiments, the sterile filtration is tangential flow filtration.

[0346] In certain embodiments, the solution is processed through a sterile filtration step in which the filter has a nominal retention range of about 0.01-0.2 μm, about 0.05-0.2 μm, about 0.1-0.2 μm, or about 0.15-0.2 μm.

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

[0348] In certain embodiments, the solution is processed by a sterile filtration step in which the filter has a nominal retention range of about 0.05, about 0.1, about 0.15, or about 0.2 μm.

[0349] In one embodiment, the solution is processed by a sterile filtration step in which the filter has a nominal retention range of about 0.2 μm.

[0350] In one embodiment, the filter has a flow capacity of about 25 to 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 2The solution is processed by a sterile filtration step with a filter capacity of 1000 s.p.m.

[0351] In one embodiment, the filter has a flow capacity of about 25 to 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 to 1000 L / m 2 The solution is processed by a sterile filtration step with a filter capacity of 1000 s.p.m.

[0352] In one embodiment, the filter has a flow rate of 25 to 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 The solution is processed by a sterile filtration step with a filter capacity of 1000 s.p.m.

[0353] In one embodiment, the filter has a flow rate of 25 to 300 L / m 2 , 50~300L / m 2 , 75~300L / m 2 , 100~300L / m 2 , 150~300L / m 2 or 250-300L / m 2 The solution is processed by a sterile filtration step with a filter capacity of 1000 s.p.m.

[0354] In one embodiment, the filter has a flow rate of 25 to 250 L / m 2 , 50~250L / m 2 , 75~250L / m 2 , 100~250L / m 2 , or 150 to 250 L / m 2 , 200~250L / m 2 The solution is processed by a sterile filtration step with a filter capacity of 1000 s.p.m.

[0355] In one embodiment, the filter has a flow rate of 25 to 100 L / m 2 , 50~100L / m 2 or 75 to 100 L / m 2 The solution is processed by a sterile filtration step with a filter capacity of 1000 s.p.m.

[0356] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0357] In some embodiments, 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 The solution is processed through a microfiltration step having a filter capacity of 1000 s.p.m.

[0358] 1.13 Final materials The polysaccharide can be finally prepared as a liquid solution. The polysaccharide can be further processed (e.g., lyophilized as a dry powder, see WO2006 / 110381). Thus, in some embodiments, the polysaccharide is a dry powder.

[0359] In some embodiments, the polysaccharide is a lyophilized cake.

[0360] 2. Use of purified polysaccharides The polysaccharide purified by the method of the present invention can be used as an antigen. Plain polysaccharide is used as an antigen in a vaccine (see 23-valent non-conjugated pneumococcal polysaccharide vaccine PNEUMOVAX).

[0361] The polysaccharides purified by the methods of the present invention can also be conjugated to a carrier protein to obtain a glycoconjugate.

[0362] 2.1. Glycoconjugates The polysaccharides purified by the methods of the present invention can be conjugated to a carrier protein to obtain a glycoconjugate.

[0363] For purposes of the present invention, the term "glycoconjugate" refers to a sugar covalently linked to a carrier protein. In one embodiment, the sugar is directly linked to the carrier protein. In a second embodiment, the sugar is linked to the carrier protein via a spacer / linker.

[0364] In general, covalent conjugation of saccharides to carriers enhances the immunogenicity of saccharides because it converts them from T cell-independent antigens to T cell-dependent antigens, thus enabling the priming of immune memory. Conjugation is particularly useful for pediatric vaccines.

[0365] Polysaccharides purified by the methods of the invention can be activated (e.g., chemically activated) to allow them to react (e.g., with a linker or directly with a carrier protein) and then incorporated into glycoconjugates, as further described herein.

[0366] The purified polysaccharide can be sized to a target molecular weight prior to conjugation, for example, by the methods disclosed in Section 1.11 above. Thus, in certain embodiments, the purified polysaccharide is sized prior to conjugation. In certain embodiments, the purified polysaccharide disclosed herein can be sized prior to conjugation to obtain oligosaccharides. Oligosaccharides have a small number of repeating units (typically 5-15 repeating units) and are typically derived by sizing (e.g., hydrolysis) of the polysaccharide.

[0367] Preferably, however, the sugar used for conjugation is a polysaccharide. High molecular weight polysaccharides can induce a specific antibody immune response due to epitopes present on their antigenic surface. Isolation and purification of high molecular weight polysaccharides are preferably contemplated for use in the conjugates of the present invention.

[0368] Thus, in some embodiments, the polysaccharide is sized but remains a polysaccharide. In some embodiments, the polysaccharide is unsized.

[0369] In some embodiments, the purified polysaccharide (either sized or unsized) prior to conjugation has a molecular weight of 5 kDa to 4,000 kDa. In other such embodiments, the purified polysaccharide has a molecular weight of 10 kDa to 4,000 kDa. In other such embodiments, the purified polysaccharide has a molecular weight of 50 kDa to 4,000 kDa. In further such embodiments, the polysaccharide has a molecular weight of 50 kDa to 3,500 kDa; 50 kDa to 3,000 kDa; 50 kDa to 2,500 kDa; 50 kDa to 2,000 kDa; 50 kDa to 1,750 kDa; 50 kDa to 1,500 kDa; 50 kDa to 1,250 kDa; 50 kDa to 1,000 kDa. a;50kDa~750kDa;50kDa~500kDa;100kDa~4,000kDa;100kDa~3,500kDa;100kDa~3, 000kDa;100kDa~2,500kDa;100kDa~2,250kDa;100kDa~2,000kDa;100kDa~1,750kDa a;100kDa~1,500kDa;100kDa~1,250kDa;100kDa~1,000kDa;100kDa~750kDa;100kDa a~500kDa;200kDa~4,000kDa;200kDa~3,500kDa;200kDa~3,000kDa;200kDa~2,500 kDa; 200 kDa to 2,250 kDa; 200 kDa to 2,000 kDa; 200 kDa to 1,750 kDa; 200 kDa to 1,500 kDa; 200 kDa to 1,250 kDa; 200 kDa to 1,000 kDa; 200 kDa to 750 kDa; or 200 kDa to 500 kDa.In further such embodiments, the polysaccharide has a molecular weight of 250 kDa to 3,500 kDa; 250 kDa to 3,000 kDa; 250 kDa to 2,500 kDa; 250 kDa to 2,000 kDa; 250 kDa to 1,750 kDa; 250 kDa to 1,500 kDa; 250 kDa to 1,250 kDa; 250 kDa to 1 ,000kDa;250kDa~750kDa;250kDa~500kDa;300kDa~4,000kDa;300kDa~3,500kDa;30 0kDa~3,000kDa;300kDa~2,500kDa;300kDa~2,250kDa;300kDa~2,000kDa;300kDa~1, 750kDa;300kDa~1,500kDa;300kDa~1,250kDa;300kDa~1,000kDa;300kDa~750kDa;3 00kDa~500kDa;500kDa~4,000kDa;500kDa~3,500kDa;500kDa~3,000kDa;500kDa~2,5 500kDa to 2,250kDa; 500kDa to 2,000kDa; 500kDa to 1,750kDa; 500kDa to 1,500kDa; 500kDa to 1,250kDa; 500kDa to 1,000kDa; 500kDa to 750kDa; or 500kDa to 600kDa.

[0370] Any number within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0371] In some embodiments, the purified polysaccharide is 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. Da, about 650 kDa, about 700 kDa, about 750 kDa, about 800 kDa, about 850 kDa, about 900 kDa, about 950 kDa, about 1000 kDa, about 1250 kDa, about 1500 kDa, about 1750 kDa, about 2000 kDa, about 2250 kDa, about 2500 kDa, about 2750 kDa, about 3000 kDa, about 3250 kDa, about 3500 kDa, about 3750 kDa or about 4,000 kDa.

[0372] In certain embodiments, the purified polysaccharide is a capsular saccharide (polysaccharide or oligosaccharide).

[0373] In some embodiments, the purified polysaccharide is a capsular polysaccharide derived from Escherichia coli. In some embodiments, the purified polysaccharide is a capsular polysaccharide derived from the Escherichia coli portion of the enterotoxigenic Escherichia coli group (EEC group), such as enterotoxigenic Escherichia coli (ETEC), pathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC)-O157:H7, or enterocytoinvasive Escherichia coli (EIEC). In some embodiments, the purified polysaccharide is a capsular polysaccharide derived from uropathogenic Escherichia coli (UPEC).

[0374] In some embodiments, the purified polysaccharide is a capsular polysaccharide derived from an Escherichia coli serotype selected from the group consisting of serotypes O157:H7, O26:H11, O111:H-, and O103:H2. In some embodiments, the purified polysaccharide is a capsular polysaccharide derived from an Escherichia coli serotype selected from the group consisting of serotypes O6:K2:H1 and O18:K1:H7. In some embodiments, the purified polysaccharide is a capsular polysaccharide derived from an Escherichia coli serotype selected from the group consisting of serotypes O45:K1, O17:K52:H18, O19:H34, and O7:K1. In some embodiments, the purified polysaccharide is a capsular polysaccharide derived from Escherichia coli serotype O104:H4. In some embodiments, the purified polysaccharide is a capsular polysaccharide derived from Escherichia coli serotype O1:K12:H7. In some embodiments, the purified polysaccharide is a capsular polysaccharide derived from Escherichia coli serotype O127:H6. In some embodiments, the purified polysaccharide is a capsular polysaccharide derived from Escherichia coli serotype O139:H28. In some embodiments, the purified polysaccharide is a capsular polysaccharide derived from Escherichia coli serotype O128:H2.

[0375] In a further embodiment, the purified polysaccharide is a capsular polysaccharide derived from Neisseria meningitidis, hi one embodiment, the purified polysaccharide is a capsular polysaccharide derived from N. meningitidis serogroup A (MenA), N. meningitidis serogroup W135 (MenW135), N. meningitidis serogroup Y (MenY), N. meningitidis serogroup X (MenX), or N. meningitidis serogroup C (MenC).

[0376] In another embodiment, the purified polysaccharide is a capsular polysaccharide derived from Klebsiella pneumoniae. In one embodiment, the source of bacterial capsular polysaccharide is selected from the group consisting of 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 O6 (O6), Klebsiella pneumoniae serogroup O7 (O7), Klebsiella pneumoniae serogroup O8 (O8), Klebsiella pneumoniae serogroup O9 (O9), Klebsiella pneumoniae serogroup O10 (O10), Klebsiella pneumoniae serogroup O11 (O11), Klebsiella pneumoniae serogroup O12 (O12), Klebsiella pneumoniae serogroup O13 (O13), Klebsiella pneumoniae serogroup O14 (O14), Klebsiella pneumoniae serogroup O15 (O15), Klebsiella pneumoniae serogroup O16 (O16), Klebsiella pneumoniae serogroup O17 (O17), Klebsiella pneumoniae serogroup O18 (O18), Klebsiella pneumoniae serogroup O19 (O19), Klebsiella pneumoniae serogroup O2ac (O2ac), Klebsiella pneumoniae serogroup O2ac (O In one embodiment, the source of bacterial capsular polysaccharide is K. pneumoniae serogroup O4 (O4), K. pneumoniae serogroup O5 (O5), K. pneumoniae serogroup O7 (O7), K. pneumoniae serogroup O8 (O8), or K. pneumoniae serogroup O9 (O9). In one embodiment, the source of bacterial capsular polysaccharide is K. pneumoniae serogroup O1 (O1). In one embodiment, the source of bacterial capsular polysaccharide is K. pneumoniae serogroup O2 (O2). In some embodiments, the source of bacterial capsular polysaccharides is K. pneumoniae serogroup O2ac (O2ac). In some embodiments, the source of bacterial capsular polysaccharides is K. pneumoniae serogroup O3 (O3). In some embodiments, the source of bacterial capsular polysaccharides is K. pneumoniae serogroup O4 (O4). In some embodiments, the source of bacterial capsular polysaccharides is K. pneumoniae serogroup O5 (O5). In some embodiments, the source of bacterial capsular polysaccharides is K. pneumoniae serogroup O7 (O7). In one embodiment, the source of bacterial capsular polysaccharides is K. pneumoniae serogroup O8 (O8). In one embodiment, the source of bacterial capsular polysaccharides is K. pneumoniae serogroup O9 (O9).

[0377] Any suitable conjugation reaction can be used, with any suitable linker where necessary, see for example WO2007116028, pages 17-22.

[0378] The purified oligosaccharides or polysaccharides described herein are chemically activated to produce saccharides that can react with carrier proteins.

[0379] In certain embodiments, the glycoconjugates are prepared using reductive amination.

[0380] Reductive amination involves two steps: (1) oxidation (activation) of purified sugars, and (2) coupling of the activated sugars with a carrier protein (e.g., CRM) to form glycoconjugates. 197 , DT, TT or PD) (see, for example, WO2015110941, WO2015110940).

[0381] As described above, sizing of the polysaccharide to a target molecular weight (MW) range can be performed prior to oxidation. Mechanical or chemical hydrolysis can be used. Chemical hydrolysis can be performed using acetic acid. In some embodiments, the size of the purified polysaccharide is reduced by mechanical homogenization.

[0382] In one embodiment, the purified polysaccharides or oligosaccharides are (a) reacting the purified polysaccharide or oligosaccharide with an oxidizing agent; (b) optionally quenching the oxidation reaction by the addition of a quenching agent; (c) mixing the activated polysaccharide or oligosaccharide of step (a) or (b) with a carrier protein; and (d) conjugating the mixed, activated polysaccharide or oligosaccharide and carrier protein to the carrier protein by a process comprising reacting with a reducing agent to form a glycoconjugate.

[0383] After the oxidation step (a), the saccharides are said to be activated and are called "activated polysaccharides or oligosaccharides".

[0384] The oxidation step (a) may involve reaction with periodate. For purposes of the present invention, the term "periodate" includes both periodate and periodic acid; the term also includes metaperiodic acid (IO4). - ) and orthoperiodate (IO6 5- ) and various salts of periodic acid (e.g., sodium periodate and potassium periodate).

[0385] In a preferred embodiment, the oxidizing agent is sodium periodate. In some embodiments, the periodate used for oxidation is metaperiodate. In some embodiments, the periodate used for oxidation is sodium metaperiodate.

[0386] The oxidation step (a) may involve reaction with a stable nitroxyl or nitroxide radical compound, such as a piperidine-N-oxy or pyrrolidine-N-oxy compound, in the presence of an oxidizing agent that selectively oxidizes primary hydroxyls of the polysaccharide or oligosaccharide to produce an activated sugar containing an aldehyde group (see WO2014097099). In one embodiment, the stable nitroxyl or nitroxide radical compound is any of those disclosed on page 3, line 14 to page 4, line 7 of WO2014097099, and the oxidizing agent is any of those disclosed on page 4, lines 8-15 of WO2014097099. In one embodiment, the stable nitroxyl or nitroxide radical compound is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and the oxidizing agent is N-chlorosuccinimide (NCS).

[0387] In one embodiment, the quenching agent is one disclosed in WO2015110941 (see page 30, lines 3-26).

[0388] In some embodiments, the reduction reaction (d) is carried out in an aqueous solvent. In some embodiments, the reduction reaction (d) is carried out in an aprotic solvent. In some embodiments, the reduction reaction (d) is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent.

[0389] In certain embodiments, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium or zinc borohydride in the presence of a Bronsted or Lewis acid, pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3, or an amine borane such as 5-ethyl-2-methylpyridine borane (PEMB). In a preferred embodiment, the reducing agent is sodium cyanoborohydride.

[0390] At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugate, which can be capped using a suitable capping agent, which in one embodiment is sodium borohydride (NaBH4).

[0391] After conjugation to a carrier protein, the glycoconjugate can be purified (enriched with respect to the amount of glyco-protein conjugate) by a variety of techniques known to those skilled in the art, including dialysis, concentration / diafiltration operations, tangential flow filtration sedimentation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration.

[0392] In certain embodiments, the glycoconjugates are prepared using cyanylation chemistry.

[0393] In one embodiment, purified polysaccharides or oligosaccharides are activated with cyanogen bromide. Activation corresponds to the cyanylation of hydroxyl groups on the polysaccharide or oligosaccharide. The activated polysaccharide or oligosaccharide is then coupled to amino groups on a carrier protein either directly or via a spacer (linker) group.

[0394] In one embodiment, purified polysaccharides or oligosaccharides are activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form cyanate esters, which are then coupled to amino groups on a carrier protein either directly or via a spacer (linker) group.

[0395] In certain embodiments, the spacer may be cystamine or cysteamine to yield a thiolated polysaccharide or oligosaccharide that 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., iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA), or succinimidyl 3-[bromoacetamido]propionate (SBAP)). Preferably, a cyanate ester (which may be generated by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH), and the amino-derivatized sugar is attached to a carrier protein (e.g., CRM) using carbodiimide (e.g., EDAC or EDC) chemistry via a carboxyl group on the protein carrier. 197 Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348 and WO 96 / 129094.

[0396] In some embodiments, glycoconjugates are prepared using bis-electrophiles 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, either via a direct route or using a bifunctional linker (see, e.g., WO2011041003).

[0397] In certain embodiments, the glycoconjugate is prepared by the method for making a glycoconjugate disclosed in WO2014027302. The resulting glycoconjugate comprises a sugar covalently conjugated to a carrier protein via a bivalent, heterobifunctional spacer (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC). Alternatively, the glycoconjugate is prepared by the method for making a glycoconjugate disclosed in WO2015121783.

[0398] Other suitable conjugation techniques use carbodiimides (e.g., EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), EDC plus sulfo-NHS, CMC (1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide), DCC (N,N'-dicyclohexylcarbodiimide), or DIC (diisopropylcarbodiimide)).

[0399] In certain embodiments, polysaccharides or oligosaccharides are conjugated to carrier proteins via linkers, e.g., bifunctional linkers. The linkers may be heterobifunctional or homobifunctional, e.g., having a reactive amino group and a reactive carboxylic acid group, two reactive amino groups, or two reactive carboxylic acid groups. The linkers may have, for example, 4 to 20, 4 to 12, or 5 to 10 carbon atoms. A possible linker is adipic acid dihydrazide (ADH). Other linkers include β-propionamide (WO 00 / 10599), nitrophenyl-ethylamine, haloalkyl halides, glycosidic bonds (U.S. Pat. Nos. 4,673,574 and 4,808,700), hexanediamine, and 6-aminocaproic acid (U.S. Pat. No. 4,459,286).

[0400] Carrier proteins A component of the glycoconjugate is a carrier protein to which a purified polysaccharide or oligosaccharide is conjugated. The terms "protein carrier" or "carrier protein" or "carrier" can be used interchangeably herein. The carrier protein should be suitable for standard conjugation procedures.

[0401] In a preferred embodiment, the carrier protein of the glycoconjugate is DT (diphtheria toxin), TT (tetanus toxoid) or fragment C of TT, CRM 197 (a non-toxic but antigenically identical variant of diphtheria toxin), other DT variants (CRM 176 , CRM 228 , CRM 45 (Uchida et al. (1973) J. Biol. Chem. 218:3838~3844), CRM9, CRM 102 , CRM 103 or CRM 107and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc. (1992); deletions or mutations of Glu-148 to Asp, Gln, or Ser and / or Ala-158 to Gly and other mutations disclosed in U.S. Pat. Nos. 4,709,017 and 4,950,740; mutations of at least one or more residues of Lys516, Lys526, Phe530, and / or Lys534 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, p ly, e.g., dPLY-GMBS (WO2004 / 081515, WO2006 / 032499) or dPLY-formol, PhtX including PhtA, PhtB, PhtD, PhtE (the sequences of PhtA, PhtB, PhtD or PhtE are disclosed in WO00 / 37105 and WO00 / 39299), and fusions of Pht proteins, e.g., PhtDE fusions, PhtBE fusions, PhtA-E (WO01 / 98334, WO03 / 054007, WO2009 / 000826), including pneumococcal pneumolysin (ply) (Kuo et al. (1995) Infect lmmun 63:2706~2713), OMPC (meningococcal outer membrane protein) (EP0372501), which is usually extracted from Neisseria meningitidis serogroup B, PorB (N.meningitidis), PD (Haemophilus influenzae protein D; see, e.g., EP 0594610B), or immunologically functional equivalents thereof, synthetic peptides (EP 0378881, EP 0427347), heat shock proteins (WO 93 / 17712, WO 94 / 03208), pertussis proteins (WO 98 / 58668, EP 0471177), cytokines, lymphokines, growth factors or hormones (WO 91 / 01146), artificial proteins containing multiple human CD4+ T cell epitopes derived from antigens from various pathogens (Falugi et al. (2001) Eur J Immunol 31:3816-3824), e.g., N19 protein (Baraldoi et al. (2004) Infect Immunol 72:4884-4887), pneumococcal surface protein PspA (WO02 / 091998), iron uptake protein (WO01 / 72337), Clostridium difficile toxin A or B (WO00 / 61761), transferrin-binding protein, pneumococcal adhesion protein (PsaA), recombinant Pseudomonas aeruginosa exotoxin A (particularly its non-toxic mutants, exotoxin A carrying 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., as described in WO 2004 / 083251), Escherichia coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa.

[0402] In a preferred embodiment, the carrier protein of the glycoconjugate is selected from the group consisting of TT, DT, DT variants (CRM 197and the like), Haemophilus influenzae (H. influenzae) protein D, PhtX, PhtD, PhtDE fusions (particularly those described in WO01 / 98334 and WO03 / 054007), detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, Clostridium difficile (C. difficile) toxin A or B, and PsaA.

[0403] 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).

[0404] In another embodiment, the carrier protein of the glycoconjugate is PD (H. influenzae protein D; see, for example, EP0594610B).

[0405] In a preferred embodiment, the purified polysaccharides or oligosaccharides are 197 Conjugate to protein. CRM 197 The protein is a non-toxic form of diphtheria toxin, but is immunologically indistinguishable from diphtheria toxin. 197 was generated by nitrosoguanidine mutagenesis of the toxigenic corynephage beta (Uchida et al. (1971) Nature New Biology 233:8-11). tox- CRM is produced by Corynebacterium diphtheriae infected with 197 The protein has the same molecular weight as diphtheria toxin but differs from it by a single base change in the structural gene (guanine to adenine). This single base change causes an amino acid substitution in the mature protein (glutamic acid to glycine), eliminating the toxicity of diphtheria toxin. 197 Proteins are safe and effective T cell-dependent carriers for sugars. 197and further details regarding its production can be found, for example, in US Pat. No. 5,614,382.

[0406] In one embodiment, the purified polysaccharides or oligosaccharides are 197 Protein or CRM 197 (See CN103495161). In one embodiment, the purified polysaccharide or oligosaccharide is conjugated to the A chain of CRM obtained via expression in recombinant E. coli. 197 (See CN103495161).

[0407] Preferably, the ratio of carrier protein to polysaccharide or oligosaccharide in the glycoconjugate is 1:5 to 5:1, such as 1:0.5 to 4:1, for example, 1:1 to 3.5:1, 1.2:1 to 3:1, 1.5:1 to 2.5:1, for example, 1:2 to 2.5:1 or 1:1 to 2:1 (w / w). In certain embodiments, 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.

[0408] After conjugation to a carrier protein, the glycoconjugate can be purified (enriched with respect to the amount of glyco-protein conjugate) by a variety of techniques known to those skilled in the art, including dialysis, concentration / diafiltration operations, tangential flow filtration sedimentation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration.

[0409] The composition may contain small amounts of free carrier. When a given carrier protein is present in a composition of the invention in both free and conjugated form, the unconjugated form preferably represents 5% or less of the total amount of carrier protein in the composition as a whole, more preferably less than 2% by weight.

[0410] 2.2 Immunogenic compositions In certain embodiments, the present invention relates to immunogenic compositions comprising any of the purified polysaccharides and / or glycoconjugates disclosed herein.

[0411] In certain embodiments, the present invention relates to an immunogenic composition comprising any of the glycoconjugates disclosed herein.

[0412] In one embodiment, the present invention relates to an immunogenic composition comprising 1 to 25 different glycoconjugates disclosed in Section 2.1.

[0413] In certain embodiments, the present invention relates to an immunogenic composition comprising 1 to 25 glycoconjugates (1-25 pneumococcal conjugates) derived from different serotypes of S. pneumoniae. In one embodiment, the present invention relates to an immunogenic composition comprising glycoconjugates derived from 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 different serotypes of S. pneumoniae. In one embodiment, the immunogenic composition comprises glycoconjugates derived from 16 or 20 different serotypes of S. pneumoniae. In certain embodiments, 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 some embodiments, the immunogenic composition is a 14-, 15-, 16-, 17-, 18-, or 19-valent pneumococcal conjugate composition. In some embodiments, the immunogenic composition is a 16-valent pneumococcal conjugate composition. In some embodiments, the immunogenic composition is a 19-valent pneumococcal conjugate composition. In some embodiments, the immunogenic composition is a 20-valent pneumococcal conjugate composition.

[0414] In one embodiment, the immunogenic composition comprises glycoconjugates derived from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.

[0415] In one embodiment, the immunogenic composition further comprises glycoconjugates derived from S. pneumoniae serotypes 1, 5 and 7F.

[0416] In certain embodiments, any of the above immunogenic compositions further comprise glycoconjugates derived from S. pneumoniae serotypes 6A and 19A.

[0417] In certain embodiments, any of the above immunogenic compositions further comprises a glycoconjugate derived from S. pneumoniae serotype 3.

[0418] In certain embodiments, any of the above immunogenic compositions further comprise glycoconjugates derived from S. pneumoniae serotypes 22F and 33F.

[0419] In certain embodiments, any of the above immunogenic compositions further comprise a glycoconjugate derived from S. pneumoniae serotypes 8, 10A, 11A, 12F, and 15B.

[0420] In certain embodiments, any of the above immunogenic compositions further comprises a glycoconjugate derived from S. pneumoniae serotype 2.

[0421] In certain embodiments, any of the above immunogenic compositions further comprises a glycoconjugate derived from S. pneumoniae serotype 9N.

[0422] In certain embodiments, any of the above immunogenic compositions further comprises a glycoconjugate derived from S. pneumoniae serotype 17F.

[0423] In certain embodiments, any of the above immunogenic compositions further comprises a glycoconjugate derived from S. pneumoniae serotype 20.

[0424] In certain embodiments, the immunogenic compositions of the invention comprise glycoconjugates derived from S. pneumoniae serotypes 8, 10A, 11A, 12F, 15B, 22F and 33F.

[0425] In certain embodiments, any of the above immunogenic compositions further comprises a glycoconjugate derived from S. pneumoniae serotype 2.

[0426] In certain embodiments, any of the above immunogenic compositions further comprises a glycoconjugate derived from S. pneumoniae serotype 9N.

[0427] In certain embodiments, any of the above immunogenic compositions further comprises a glycoconjugate derived from S. pneumoniae serotype 17F.

[0428] In certain embodiments, any of the above immunogenic compositions further comprises a glycoconjugate derived from S. pneumoniae serotype 20.

[0429] However, in a preferred embodiment, the saccharides are each individually conjugated to different molecules of the protein carrier (each molecule of the protein carrier has only one type of saccharide conjugated to it). In said embodiment, the capsular saccharides are said to be individually conjugated to the carrier protein. Preferably, all saccharide conjugates of the immunogenic composition are individually conjugated to the carrier protein.

[0430] In an embodiment of any of the above immunogenic compositions, the glycoconjugate derived from S. pneumoniae serotype 22F is administered in a CRM 197 In embodiments of any of the above immunogenic compositions, the glycoconjugate derived from S. pneumoniae serotype 33F is conjugated to a CRM 197In embodiments of any of the above immunogenic compositions, the glycoconjugate derived from S. pneumoniae serotype 15B is conjugated to a CRM 197 In embodiments of any of the above immunogenic compositions, the glycoconjugate derived from S. pneumoniae serotype 12F is conjugated to a CRM 197 In embodiments of any of the above immunogenic compositions, the glycoconjugate derived from S. pneumoniae serotype 10A is conjugated to a CRM 197 In embodiments of any of the above immunogenic compositions, the glycoconjugate derived from S. pneumoniae serotype 11A is conjugated to a CRM 197 In embodiments of any of the above immunogenic compositions, the glycoconjugate derived from S. pneumoniae serotype 8 is conjugated to a CRM 197 In embodiments of any of the above immunogenic compositions, glycoconjugates derived from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F are conjugated to CRM 197 In embodiments of any of the above immunogenic compositions, glycoconjugates derived from S. pneumoniae serotypes 1, 5 and 7F are conjugated to a CRM 197 In embodiments of any of the above immunogenic compositions, glycoconjugates derived from S. pneumoniae serotypes 6A and 19A are conjugated to CRM 197 In embodiments of any of the above immunogenic compositions, the glycoconjugate derived from S. pneumoniae serotype 3 is conjugated to a CRM 197 Conjugate to.

[0431] In one embodiment, the glycoconjugates of any of the above immunogenic compositions are all individually administered in a CRM 197 Conjugate to.

[0432] In certain embodiments, glycoconjugates derived from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and / or 23F of any of the above immunogenic compositions are individually conjugated to PD.

[0433] In certain embodiments, the glycoconjugate derived from S. pneumoniae serotype 18C of any of the above immunogenic compositions is conjugated to TT.

[0434] In certain embodiments, the glycoconjugate derived from S. pneumoniae serotype 19F of any of the above immunogenic compositions is conjugated to DT.

[0435] In certain embodiments, glycoconjugates derived from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and / or 23F of any of the above immunogenic compositions are individually conjugated to PD, glycoconjugates derived from S. pneumoniae serotype 18C are conjugated to TT, and glycoconjugates derived from S. pneumoniae serotype 19F are conjugated to DT.

[0436] In one embodiment, the immunogenic composition comprises 8 to 20 different serotypes of S. pneumoniae.

[0437] In certain embodiments, the present invention relates to an immunogenic composition comprising one to five glycoconjugates (one to five meningococcal conjugates) derived from different N. meningitidis serogroups. In one embodiment, the present invention relates to an immunogenic composition comprising glycoconjugates derived from one, two, three, four, or five different N. meningitidis serogroups. In one embodiment, the immunogenic composition comprises four or five different N. meningitidis species. In certain embodiments, the immunogenic composition is a mono-, bi-, tri-, tetra-, or pentavalent meningococcal conjugate composition. In certain embodiments, the immunogenic composition is a bivalent meningococcal conjugate composition. In certain embodiments, the immunogenic composition is a tetravalent meningococcal conjugate composition. In certain embodiments, the immunogenic composition is a pentavalent meningococcal conjugate composition.

[0438] In an embodiment, the immunogenic composition comprises conjugated N. meningitidis serogroup Y capsular saccharide (MenY), and / or conjugated N. meningitidis serogroup C capsular saccharide (MenC).

[0439] In an embodiment, the immunogenic composition comprises a conjugated N. meningitidis serogroup A capsular saccharide (MenA), a conjugated N. meningitidis serogroup W135 capsular saccharide (MenW135), a conjugated N. meningitidis serogroup Y capsular saccharide (MenY), and / or a conjugated N. meningitidis serogroup C capsular saccharide (MenC).

[0440] In an embodiment, the immunogenic composition comprises conjugated N. meningitidis serogroup W135 capsular saccharide (MenW135), conjugated N. meningitidis serogroup Y capsular saccharide (MenY), and / or conjugated N. meningitidis serogroup C capsular saccharide (MenC).

[0441] In an embodiment the immunogenic composition comprises a conjugated N. meningitidis serogroup A capsular saccharide (MenA), a conjugated N. meningitidis serogroup W135 capsular saccharide (MenW135), a conjugated N. meningitidis serogroup Y capsular saccharide (MenY), a conjugated N. meningitidis serogroup C capsular saccharide (MenC) and / or a conjugated N. meningitidis serogroup X capsular saccharide (MenX).

[0442] In some embodiments, the immunogenic compositions disclosed herein may further comprise at least one, two, or three adjuvants. In some embodiments, the immunogenic compositions disclosed herein may further comprise one adjuvant. The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. The antigen may act primarily as a delivery system, primarily as an immunomodulator, or have both potent properties. Suitable adjuvants include those suitable for use in mammals, including humans.

[0443] 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.

[0444] In some embodiments, the immunogenic compositions disclosed herein comprise an aluminum salt (alum) (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide) as an adjuvant. In preferred embodiments, the immunogenic compositions disclosed herein comprise aluminum phosphate or aluminum hydroxide as an adjuvant.

[0445] Additional exemplary adjuvants for enhancing the effectiveness of the immunogenic compositions disclosed herein include, but are not limited to, (1) SAF containing, for example, (a) 10% squalane, 0.4% Tween 80, 5% Pluronic blocking polymer L121, and thr-MDP microfluidized to a submicron emulsion or vortexed to generate a larger particle size emulsion, and (b) RIBI™ Adjuvant System (RAS) (Ribi™) 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 (DETOX™). (2) oil-in-water emulsion formulations (with or without muramyl peptides (see below) or other specific immunostimulants such as bacterial cell wall components), such as (Immunochem, Hamilton, MT); (3) QS21, STIMULON™ (Cambridge Bioscience, Worcester, MA), ABISCO® (Isconova, Sweden), or ISCOMATRIX® (Commonwealth Serum), which can be used. (3) saponin adjuvants such as saponin adjuvants, such as saponin adjuvants (S. Laboratories, Australia) or particles produced therefrom, such as ISCOMs (immunostimulating complexes), which may contain no additional detergents (see, e.g., WO 00 / 07621); (4) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (see, e.g., WO 99 / 44636)), interferons (e.g., gamma interferon), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), and the like;(5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL), optionally in the substantial absence of alum, when used with pneumococcal saccharides (see, e.g., WO 00 / 56358) (see, e.g., GB-2220221, EP 0689454); (6) combinations of 3dMPL with, for example, QS21 and / or oil-in-water emulsions (see, e.g., EP 0835318, EP 0735898, EP 0761231); (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 / 212 (9) saponin and immunostimulatory oligonucleotides (e.g., CpG oligonucleotides) (e.g., WO 00 / 62800); (10) particles of immunostimulants and metal salts (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. Examples of muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE).

[0446] In an embodiment of the present invention, the immunogenic compositions disclosed herein comprise a CpG oligonucleotide as an adjuvant.

[0447] The immunogenic compositions can be formulated in liquid form (i.e., solution or suspension) or lyophilized form. Advantageously, liquid formulations can be administered directly from their packaging, and thus are ideal for injection, without the need for reconstitution in an aqueous medium that would otherwise be required for lyophilized compositions of the invention.

[0448] The immunogenic compositions of the present disclosure can be formulated using art-recognized methods. For example, individual polysaccharides and / or conjugates can be formulated with a physiologically acceptable vehicle to prepare the composition. Examples of such vehicles include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and dextrose solutions.

[0449] The present disclosure provides immunogenic compositions comprising any of the polysaccharide or sugar conjugate combinations disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0450] In certain embodiments, the immunogenic compositions of the present disclosure are in liquid form, preferably in aqueous liquid form.

[0451] The immunogenic compositions of the present disclosure may include one or more of a buffering agent, 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 chelating agent, or any combination thereof.

[0452] In one embodiment, the immunogenic composition of the present disclosure comprises a buffer. In one embodiment, the buffer has a pKa of about 3.5 to about 7.5. In some embodiments, the buffer is a phosphate buffer, a succinate buffer, a histidine buffer, or a citrate buffer. In a specific embodiment, the buffer is a succinate buffer at a final concentration of 1 mM to 10 mM. In one specific embodiment, the final concentration of the succinate buffer is about 5 mM.

[0453] In some embodiments, the immunogenic composition of the present disclosure 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 one specific embodiment, the salt is sodium chloride. In one specific embodiment, the immunogenic composition of the present disclosure comprises 150 mM sodium chloride.

[0454] In some embodiments, the immunogenic composition of the present disclosure comprises a surfactant selected from the group consisting of polysorbate 20 (TWEEN™ 20), polysorbate 40 (TWEEN™ 40), polysorbate 60 (TWEEN™ 60), polysorbate 65 (TWEEN™ 65), polysorbate 80 (TWEEN™ 80), polysorbate 85 (TWEEN™ 85), TRITON™ N-101, TRITON™ X-100, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol H15), polyoxyethylene-35-ricinoleate (CREMOPHOR® EL), soy lecithin, and poloxamer. In one particular embodiment, the surfactant is polysorbate 80. In some such embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.0001% to 10% (w / w) polysorbate 80. In some such embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.001% to 1% (w / w) polysorbate 80. In some such embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.01% to 1% (w / w) polysorbate 80. 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% (w / w) polysorbate 80. In another embodiment, the final concentration of polysorbate 80 in the formulation is 1% polysorbate 80 by weight (w / w).

[0455] In one particular embodiment, the surfactant is polysorbate 20. In some such embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.0001% to 10% (w / w) polysorbate 20. In some such embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.001% to 1% (w / w) polysorbate 20. In some such embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.01% to 1% (w / w) polysorbate 20. 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% (w / w) polysorbate 20. In another embodiment, the final concentration of polysorbate 20 in the formulation is 1% by weight (w / w) polysorbate 20.

[0456] In one particular embodiment, the surfactant is polysorbate 40. In some such embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.0001% to 10% (w / w) polysorbate 40. In some such embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.001% to 1% (w / w) polysorbate 40. In some such embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.01% to 1% (w / w) polysorbate 40. 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% (w / w) polysorbate 40. In another embodiment, the final concentration of polysorbate 40 in the formulation is 1% polysorbate 40 by weight (w / w).

[0457] In one particular embodiment, the surfactant is polysorbate 60. In some such embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.0001% to 10% (w / w) polysorbate 60. In some such embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.001% to 1% (w / w) polysorbate 60. In some such embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.01% to 1% (w / w) polysorbate 60. 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% (w / w) polysorbate 60. In another embodiment, the final concentration of polysorbate 60 in the formulation is 1% polysorbate 60 by weight (w / w).

[0458] In one particular embodiment, the surfactant is polysorbate 65. In some such embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.0001% to 10% (w / w) polysorbate 65. In some such embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.001% to 1% (w / w) polysorbate 65. In some such embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.01% to 1% (w / w) polysorbate 65. 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% (w / w) polysorbate 65. In another embodiment, the final concentration of polysorbate 65 in the formulation is 1% polysorbate 65 by weight (w / w).

[0459] In one particular embodiment, the surfactant is polysorbate 85. In some such embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.0001% to 10% (w / w) polysorbate 85. In some such embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.001% to 1% (w / w) polysorbate 85. In some such embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.01% to 1% (w / w) polysorbate 85. 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% (w / w) polysorbate 85. In another embodiment, the final concentration of polysorbate 85 in the formulation is 1% polysorbate 85 by weight (w / w).

[0460] 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, and even more preferably a pH of 5.8 to 6.0.

[0461] In one embodiment, the present disclosure provides a container filled with any 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 fermentor, a bioreactor, a bag, a jar, an ampoule, a cartridge, and a disposable pen. In certain embodiments, the container is siliconized.

[0462] In some embodiments, the containers of the present disclosure are made of glass, metal (e.g., steel, stainless steel, aluminum, etc.), and / or polymer (e.g., thermoplastic, elastomer, thermoplastic-elastomer). In some embodiments, the containers of the present disclosure are made of glass.

[0463] In one embodiment, the present disclosure provides a syringe filled with any of the immunogenic compositions disclosed herein. In certain embodiments, the syringe is siliconized and / or made of glass.

[0464] 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, and even more preferably about 0.5 mL.

[0465] 2.3 Use as an antigen The polysaccharides purified by the methods of the present invention and the conjugates disclosed herein can be used as antigens, for example, they can be part of a vaccine.

[0466] Thus, in one embodiment, the polysaccharide purified by the method of the invention or the glycoconjugate obtained using said polysaccharide is for use in generating an immune response in a subject. 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.

[0467] In one embodiment, the polysaccharide purified by the method of the present invention, the glycoconjugate obtained using said polysaccharide, or the immunogenic composition disclosed herein is for use in a vaccine.

[0468] In certain embodiments, the polysaccharide purified by the methods of the invention, the glycoconjugate obtained using said polysaccharide, or the immunogenic composition disclosed herein is for use as a medicament.

[0469] The immunogenic compositions described herein can be used in a variety of therapeutic or prophylactic methods for preventing, treating, or ameliorating a bacterial infection, disease, or condition in a subject. In particular, the immunogenic compositions described herein can be used to prevent, treat, or ameliorate a Streptococcus pneumoniae (S. pneumoniae), Staphylococcus aureus (S. aureus), Enterococcus faecalis (E. faecalis), Haemophilus influenzae type b, Escherichia coli (E. coli), Neisseria meningitidis, S. agalactiae, or Klebsiella pneumoniae infection, disease, or condition in a subject.

[0470] Thus, in one aspect, the present disclosure provides a method of preventing, treating, or ameliorating an infection, disease, or condition associated with Streptococcus pneumoniae (S. pneumoniae), Staphylococcus aureus (S. aureus), Enterococcus faecalis (E. faecalis), Haemophilus influenzae type b, Escherichia coli (E. coli), Neisseria meningitidis, S. agalactiae, or Klebsiella pneumoniae in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present disclosure (particularly an immunogenic composition comprising the corresponding polysaccharide or sugar conjugate thereof).

[0471] In certain embodiments, the present disclosure provides methods for inducing an immune response to Streptococcus pneumoniae (S. pneumoniae), Staphylococcus aureus (S. aureus), Enterococcus faecalis (E. faecalis), Haemophilus influenzae type b, Escherichia coli (E. coli), Neisseria meningitidis, S. agalactiae, or Klebsiella pneumoniae in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present disclosure (particularly an immunogenic composition comprising the corresponding polysaccharide or sugar conjugate thereof).

[0472] In some embodiments, the immunogenic compositions disclosed herein are for use as vaccines. In such embodiments, the immunogenic compositions described herein can be used to prevent infection in a subject with Streptococcus pneumoniae (S. pneumoniae), Staphylococcus aureus (S. aureus), Enterococcus faecalis (E. faecalis), Haemophilus influenzae type b, Escherichia coli (E. coli), Neisseria meningitidis, or S. agalactiae. Thus, in one aspect, the present invention provides a method of preventing infection by Streptococcus pneumoniae (S. pneumoniae), Staphylococcus aureus (S. aureus), Enterococcus faecalis (E. faecalis), Haemophilus influenzae type b, Escherichia coli (E. coli), Neisseria meningitidis, S. agalactiae, or Klebsiella pneumoniae in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present disclosure.

[0473] 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.

[0474] The immunogenic compositions of the present disclosure can be used to protect or treat humans susceptible to Streptococcus pneumoniae (S. pneumoniae), Staphylococcus aureus (S. aureus), Enterococcus faecalis (E. faecalis), Haemophilus influenzae type b, Escherichia coli (E. coli), Neisseria meningitidis, S. agalactiae, or Klebsiella pneumoniae infection by administering the immunogenic compositions via systemic or mucosal routes. In some embodiments, the immunogenic compositions disclosed herein are administered intramuscularly, intraperitoneally, intradermally, or subcutaneously. In some embodiments, the immunogenic compositions disclosed herein are administered by intramuscular, intraperitoneal, intradermal, or subcutaneous injection. In some embodiments, the immunogenic compositions disclosed herein are administered by intramuscular or subcutaneous injection.

[0475] In some cases, only one dose of an immunogenic composition according to the present disclosure is required, although under some circumstances, such as in more highly immunodeficient states, a second, third, or fourth dose may be given. After the initial vaccination, the subject may receive one or several booster immunizations, spaced well apart.

[0476] In some embodiments, the vaccination schedule for an immunogenic composition according to the present disclosure is a single dose.

[0477] In some embodiments, the vaccination schedule with an immunogenic composition according to the present disclosure is a multiple dose schedule.

[0478] 3. Sugars derived from E. coli In one embodiment, the sugar is produced in a recombinant Gram-negative bacterium. In one embodiment, the sugar is produced in a recombinant E. coli cell. In one embodiment, the sugar is produced in a recombinant Salmonella cell. 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 Examples of suitable bacterial strains include E. coli GAR2401, Salmonella enterica serovar Typhimurium strain LT2, E. coli GAR2401, Salmonella enterica serovar Enteritidis CVD1943, Salmonella enterica serovar Typhimurium strain CVD1925, Salmonella enterica serovar Paratyphi A CVD1902, and Shigella flexneri CVD1208S. In one embodiment, the bacterium is not E. coli GAR2401. This genetic approach to sugar production allows for efficient production of O polysaccharides and O antigen molecules as vaccine components.

[0479] As used herein, the term "wzz protein" refers to, for example, wzzB, wzz, wzz SF , wzz ST , fepE, wzz fepE, wzz1, and wzz2. GenBank accession numbers for 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. GenBank accession numbers for G7 and Bi316-41 wzz gene sequences are U39305 and U39306, respectively. Additional GenBank accession numbers for exemplary wzz gene sequences are: NP_459581 for Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 FepE; AIG66859 for E. coli O157:H7 strain EDL933 FepE; NP_461024 for Salmonella enterica subsp. enterica 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 preferred embodiments, the wzz family proteins are selected from the group consisting of wzzB, wzz, and wzz. SF , wzz ST , fepE, wzz fepE , wzz1 and wzz2, most preferably wzzB, and more preferably fepE.

[0480] Exemplary wzzB sequences include: >O25b 2401 WzzB MRVENNNVSGQNHDPEQIDLIDLLVQLWRGKMTIIISVIVAIALAIGYLAVAKEKWTSTAIITQPDVGQIAGYNNAMNVIYGQAAPKVSDLQETLIGRFSSAFSALAETLDNQEEPEKLTIEPSVKNQQLPLTVSYVGQTAEGAQMKLAQYIQQVDDKVNQELEKDLKDNIALGRKNLQDSLRTQEVVAQEQKDLRIRQIQEALQYANQEQVTKPQVQQTEDVTQDTLFLLGSEALESMIKHEATRPLVFSSNYYQTRQNLLDIESLKVDDLDIHAYRYVMKPTLPIRRDSPKKAITLILAVLLGGMVGAGIVLGRNALRNYNAK(SEQ ID NO: 20) >O25a:K5:H1 WzzB MRVENNNVSGQNNDPEQIDLIDLLVQLWRGKMTIIISVIVAIALAIGYLAVAKEKWTSTAIITQPDVGQIAGYNNAMNVIYGQAAPKVSDLQETLIGRFSSAFSALAETLDNQDEPEKLTIEPSVKNQQLPLTVSYVGQTAEGAQMKLAQYIQQVDDKVNQELEKDLKDNIALGRKNLQDSLRTQEVVAQEQKDLRIRQIQEALQYANQAQVTKPQIQQTGEDITQDTLFLLGSEALESMIKHEATRPLVFSPNYYQTRQNLLDIESLKVDDLDIHAYRYVMKPTLPIRRDSPKKAITLILAVLLGGMVGAGIVLGRNALRNYNAK(SEQ ID NO: 21) >O25a ETEC ATCC WzzB MRVENNNVSGQNHDPEQIDLIDLLVQLWRGKMTIIISVVVAIALAIGYLAVAKEKWTSTAIITQPDVGQIAGYNNAMNVIYGQAAPKVSDLQETLIGRFSFAFSALAETLDNQKEPEKLTIEPSVKNQQLPLTVSYVGQTAEDAQMKLAQYIQQVDDKVNQELEKDL KDNLALGRKNLQDSLRTQEVVAQEQKDLRIRQIQEALQYANQAQVTKPQIQQTGEDITQDTLFLLGSEALESMIKHEATRPLVFSPNYYQTRQNLLDIENLKVDDLDIHAYRYVMKPTLPIRRDSPKKAITLILAVLLGGMVGAGIVLGRNALRNYNSK (SEQ ID NO: 22) >K12 W3110 WzzB MRVENNNVSGQNHDPEQIDLIDLLVQLWRGKMTIIISVIVAIALAIGYLAVAKEKWTSTAIITQPDVGQIAGYNNAMNVIYGQAAPKVSDLQETLIGRFSSAFSALAETLDNQEEREKLTIEPSVKNQQLPLTVSYVGQTAEGAQMKLAQYIQQVDDKVNQELEKDL KDNIALGRKNLQDSLRTQEVVAQEQKDLRIRQIQEALQYANQAQVTKPQIQQTGEDITQDTLFLLGSEALESMIKHEATRPLVFSPNYYQTRQNLLDIESLKVDDLDIHAYRYVMKPMLPIRRDSPKKAITLILAVLLGGMVGAGIVLGRNALRNYNAK (SEQ ID NO: 23) >Salmonella LT2 WzzB MTVDSNTSSGRGNDPEQIDLIELLLQLWRGKMTIIVAVIIAILLAVGYLMIAKEKWTSTAIITQPDAAQVATYTNALNVLYGGNAPKISEVQANFISRFSSAFSALSEVLDNQKEREKLTIEQSVKGQALPLSVSYVSTTAEGAQRRLAEYIQQVDEEVAKELEVDLKDNITLQTKTLQESLETQEVVAQEQKDLRIKQIEEALRYADEAKITQPQIQQTQDVTQDTMFLLGSDALKSMIQNEATRPLVFSPAYYQTKQTLLDIKNLKVTADTVHVYRYVMKPTLPVRRDSPKTAITLVLAVLLGGMIGAGIVLGRNALRSYKPKAL (SEQ ID NO: 24) Examples include:

[0481] Exemplary FepE sequences include: >O25b GAR2401 FepE MSSLNIKQGSDAHFPDYPLASPSNNEIDLLNLISVLWRAKKTVMAVVFAFACAGLLISFILPQKWTSAAVVTPPEPVQWQELEKSFTKLRVLDLDIKIDRTEAFNLFIKKFQSVSLLEEYLRSSPYVMDQLKEAKIDELDLHRAIVALSEKMKAVDDNASKKKDEPSLYTSWTLSFTAPTSEEAQTVLSGYI DYISTLVVKESLENVRNKLEIKTQFEKEKLAQDRIKTKNQLDANIQRLNYSLDIANAAGIKKPVYSNGQAVKDDPDFSISLGADGIERKLEIEKAVTDVAELNGELRNRQYLVEQLTKAHVNDVNFTPFKYQLSPSLPVKKDGPGKAIIVILSALIGGMVACGGVLLRYAMASRKQDAMMADHLV (Sequence number 15) >O25a:K5:H1 FepE MSSLNIKQGSEAHFPEYPLASPSNNEIDLLNLIEVLWRAKKTVMAVVFAFACAGLLISFILPQKWTSAAVVTPPEPVQWQELEKTFTKLRVLDLDIKIDRTEAFNLFIKKFQSVSLLEEYLRSSPYVMDQLKEAKIDPLDLHRAIVALSEKMKAVDDNASKKKDESALYTSWTLSFTAPTSEEAQKVLAGYIDYISALVVKESIENVRNKLEIKTQFEKEKLAQDRIKTKNQLDANIQRLNYSLDIANAAGIKKPVYSNGQAVKDDPDFSISLGADGIERKLEIEKAVTDVAELNGELRNRQYLVEQLTKTNINDVNFTPFKYQLRPSLPVKKDGQGKAIIVILSALVGGMVACGGVLLRHAMASRKQDAMMADHLV(SEQ ID NO: 16) >O25a ETEC ATCC FepE MSSLNIKQGSDAHFPDYPLASPSNNEIDLLNLISVLWRAKKTVMAVVFAFACAGLLISFILPQKWTSAAVVTPPEPVQWQELEKSFTKLRVLDLDIKIDRTEAFNLFIKKFQSVSLLEEYLRSSPYVMDQLKEAKIDELDLHRAIVALSEKMKAVDDNASKKKDEPSLYTSWTLSFTAPTSEEAQTVLSGYIDYISTLVVKESLENVRNKLEIKTQFEKEKLAQDRIKTKNQLDANIQRLNYSLDIANAAGIKKPVYSNGQAVKDDPDFSISLGADGIERKLEIEKAVTDVAELNGELRNRQYLVEQLTKAHVNDVNFTPFKYQLSPSLPVKKDGPGKAIIVILSALIGGMVACGGVLLRYAMASRKQDAMMADHLV(SEQ ID NO: 17) >O157 FepE MSSLNIKQGSDAHFPDYPLASPSNNEIDLLNLISVLWRAKKTVMAVVFAFACAGLLISFILPQKWTSAAVVTPPEPVQWQELEKTFTKLRVLDLDIKIDRTEAFNLFIKKFQSVSLLEEYLRSSPYVMDQLKEAKIDELDLHRAIVALSEKMKAVDDNASKKKDEPSLYTSWTLSFTAPTSEEAQTVLSGYIDYISALVVKESIENVRNKLEIKTQFEKEKLAQDRIKMKNQLDANIQRLNYSLDIANAAGIKKPVYSNGQAVKDDPDFSISLGADGIERKLEIEKAVTDVAELNGELRNRQYLVEQLTKANINDVNFTPFKYQLSPSLPVKKDGPGKAIIVILSALIGGMVACGSVLLRYAMASRKQDAMMADHLV (SEQ ID NO: 18) >Salmonella LT2 FepE MPSLNVKQEKNQSFAGYSLPPANSHEIDLFSLIEVLWQAKRRILATVFAFACVGLLLSFLLPQKWTSQAIVTPAESVQWQGLERTLTALRVLDMEVSVDRGSVFNLFIKKFSSPSLLEEYLRSSPYVMDQLKGAQIDEQDLHRAIVLLSEKMKAVDSNVGKKNETSLFTSWTLSFTAPTREEAQKVLAGYIQYISDIVVKETLENIRNQLEIKTRYEQEKLAMDRVRLKNQLDANIQRLHYSLEIANAAGIKRPVYSNGQAVKDDPDFSISLGADGISRKLEIEKGVTDVAEIDGDLRNRQYHVEQLAAMNVSDVKFTPFKYQLSPSLPVKKDGPGKAIIIILAALIGGMMACGGVLLRHAMVSRKMENALAIDERLV (SEQ ID NO: 19) Examples include:

[0482] In some embodiments, modified sugars (modified relative to the corresponding wild-type sugar) can be produced in Gram-negative bacteria by expressing (not necessarily overexpressing) a wzz family protein (e.g., fepE) from Gram-negative bacteria and / or by switching off (i.e., suppressing, deleting, removing) a second wzz gene (e.g., wzzB) to produce high molecular weight sugars, such as lipopolysaccharides, containing intermediate or long O-antigen chains. For example, modified sugars can be produced by expressing (not necessarily overexpressing) wzz2 and switching off wzz1. Alternatively, modified sugars can be produced by expressing (not necessarily overexpressing) wzzfepE and switching off wzzB. In another embodiment, modified sugars can be produced by expressing (not necessarily overexpressing) wzzB but switching off wzzfepE. In another embodiment, modified sugars can be produced by expressing fepE. Preferably, the wzz family protein is derived from a strain that is heterologous to the host cell.

[0483] In one aspect, the invention relates to saccharides produced by expressing a wzz family protein, preferably fepE, in Gram-negative bacteria to produce high molecular weight saccharides containing medium or long O antigen chains with an increased number of repeating units of at least 1, 2, 3, 4, or 5 compared to the corresponding wild-type O polysaccharide. In one aspect, the invention relates to saccharides produced by expressing (but not necessarily overexpressing) a wzz family protein (e.g., wzzB) in Gram-negative bacteria to produce high molecular weight saccharides containing medium or long O antigen chains with an increased number of repeating units of at least 1, 2, 3, 4, or 5 compared to the corresponding wild-type O antigen. See the description of O polysaccharides and O antigens below for additional exemplary saccharides with an increased number of repeating units compared to the corresponding wild-type saccharide. Desired chain lengths are those that result in improved or maximal immunogenicity in the context of a given vaccine construct.

[0484] 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, 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117 , 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, and more than 100 repeat units. Preferably, the saccharide comprises an increase of 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 saccharide length are known in the art. Such methods include nuclear magnetic resonance, mass spectrometry, and size exclusion chromatography.

[0485] In a preferred embodiment, the present invention relates to saccharides produced in recombinant E. coli host cells in which the endogenous wzz O-antigen length regulator gene (e.g., wzzB) has been deleted and replaced with a (second) wzz gene (e.g., Salmonella fepE) from a Gram-negative bacterium heterologous to the recombinant E. coli host cell, producing high molecular weight saccharides, such as lipopolysaccharides, containing medium or long O-antigen chains. In some embodiments, the recombinant E. coli host cell contains a wzz gene from Salmonella, preferably Salmonella enterica.

[0486] In one embodiment, the host cell comprises a heterologous gene for a wzz family protein as a stably maintained plasmid vector. In another embodiment, the host cell comprises a heterologous gene for a wzz family protein as an integrated gene in the chromosomal DNA of the host cell. Methods for stable expression of plasmid vectors in E. coli host cells and for integrating heterologous genes into the chromosome of E. coli host cells are known in the art. In one embodiment, the host cell comprises a heterologous gene for an O antigen as a stably maintained plasmid vector. In another embodiment, the host cell comprises a heterologous gene for an O antigen as an integrated gene in the chromosomal DNA of the host cell. Methods for stable expression of plasmid vectors in E. coli host cells and Salmonella host cells are known in the art. Methods for integrating heterologous genes into the chromosome of E. coli host cells and Salmonella host cells are known in the art.

[0487] In one aspect, the recombinant host cells are cultured in a medium containing a carbon source. Carbon sources for culturing E. coli are known in the art. Exemplary carbon sources include, but are not limited to, sugar alcohols, polyols, aldol sugars, or keto sugars, such as arabinose, cellobiose, fructose, glucose, glycerol, inositol, lactose, maltose, mannitol, mannose, rhamnose, raffinose, sorbitol, sorbose, sucrose, trehalose, pyruvate, succinate, and methylamine. In a preferred embodiment, the medium contains glucose. In some embodiments, the medium contains a polyol or aldol sugar, such as mannitol, inositol, sorbose, glycerol, sorbitol, lactose, and arabinose, as a carbon source. All carbon sources may be added to the medium before the start of cultivation, or may be added in portions or continuously during cultivation.

[0488] An exemplary culture medium for recombinant host cells comprises elements selected from any one of KH2PO4, K2HPO4, (NH4)2SO4, sodium citrate, Na2SO4, aspartic acid, glucose, MgSO4, FeSO4-7H2O, Na2MoO4-2H2O, H3BO3, CoCl2-6H2O, CuCl2-2H2O, MnCl2-4H2O, ZnCl2, and CaCl2-2H2O. Preferably, the medium comprises KH2PO4, K2HPO4, (NH4)2SO4, sodium citrate, Na2SO4, aspartic acid, glucose, MgSO4, FeSO4-7H2O, Na2MoO4-2H2O, H3BO3, CoCl2-6H2O, CuCl2-2H2O, MnCl2-4H2O, ZnCl2, and CaCl2-2H2O.

[0489] As used herein, a medium may be solid or liquid, synthetic (i.e., artificial) or natural, and may contain sufficient nutrients for the cultivation of recombinant host cells. Preferably, the medium is a liquid medium.

[0490] In some embodiments, the medium may further comprise suitable inorganic salts. In some embodiments, the medium may further comprise micronutrients. In some embodiments, the medium may further comprise growth factors. In some embodiments, the medium may further comprise an additional carbon source. In some embodiments, the medium may further comprise suitable inorganic salts, micronutrients, growth factors, and additional carbon sources. Suitable inorganic salts, micronutrients, growth factors, and supplemental carbon sources for culturing E. coli are known in the art.

[0491] In some embodiments, the medium may optionally contain additional components such as peptone, NZ amine, soybean enzymatic hydrolysate, additional yeast extract, malt extract, supplemental carbon sources, and various vitamins. In some embodiments, the medium does not contain such additional components such as peptone, NZ amine, soybean enzymatic hydrolysate, additional yeast extract, malt extract, supplemental carbon sources, and various vitamins.

[0492] 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.

[0493] In some embodiments, the medium further comprises a nitrogen source. Suitable nitrogen sources for culturing E. coli are known in the art. Examples of suitable nitrogen sources include, but are not limited to, ammonia, including ammonia gas and aqueous ammonia; ammonium salts of inorganic or organic acids, such as ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate, and ammonium acetate; urea; nitrate or nitrite, and other nitrogen-containing materials, including amino acids, meat extract, peptone, fish meal, fish hydrolysate, corn infusion, casein hydrolysate, soybean meal hydrolysate, yeast extract, dry yeast, ethanol-yeast distillate, soybean flour, cottonseed flour, and the like, in pure or crude preparations.

[0494] In some embodiments, the medium comprises inorganic salts. Examples of suitable inorganic salts include, but are not limited to, salts of potassium, calcium, sodium, magnesium, manganese, iron, cobalt, zinc, copper, molybdenum, tungsten, and other trace elements, and phosphate.

[0495] In some embodiments, the medium comprises suitable growth factors. Examples of suitable 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-thioctic acid, folic acid, vitamin B, as pure or partially purified compounds or present in natural sources. 12, other vitamins, amino acids such as cysteine ​​and hydroxyproline, bases such as adenine, uracil, guanine, thymine and cytosine, sodium thiosulfate, p- or r-aminobenzoic acid, niacinamide, nitriloacetate, etc. Those skilled in the art can empirically determine the amount according to methods and techniques known in the art.

[0496] In another embodiment, the modified sugars described herein (as compared to the corresponding wild-type sugars) are synthetically produced, e.g., in vitro. Synthetic production or synthesis of sugars can facilitate avoiding costly and time-intensive production processes. In one embodiment, the sugars are synthesized, e.g., by using a sequential glycosylation strategy or a combination of a sequential glycosylation strategy and a [3+2] block synthesis strategy from a suitably protected monosaccharide intermediate. For example, thiol glycosides and glycosyl trichloroacetimidate derivatives can be used as glycosyl donors in glycosylation. In one embodiment, the sugars synthesized in vitro have the same structure as sugars produced by recombinant means, such as engineering the wzz family proteins described above.

[0497] The sugars produced (by recombinant or synthetic means) can be, for example, from the following Escherichia coli (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 (180 / C3 strain)), 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 and O45rel), O46, O48, O49, O50, O51, O52, O53, O54, O55, O56, O57, O58, O59, O60, O61, O62, 62D1, O63, O64, O65, O66, O68, O69, O70, O71, O73 (e.g., O73 (73-1 strain)), O74, O75, O76, O77, O78, O79, O80, O81, O82, O83, O84, O85, O86, O87, O88, O89, O90, O91, O92, O93, O95, O96, O97, O98, O99, O100, O101, O102, O103, O104, O105, O106, O107, O108, O109, O110, O111, O11 2, O113, O114, O115, O116, O117, O118, O119, O120, O121, O123, O124, O1 25, O126, O127, O128, O129, O130, O131, O132, O133, O134, O135, O136, O 137, O138, O139, O140, O141, O142, O143, O144, O145, O146, O147, O148, comprising a structure derived from any E. coli serotype, including any one of O149, O150, O151, O152, O153, O154, O155, O156, O157, O158, 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;

[0498] Individual polysaccharides are typically purified (enriched with respect to the amount of polysaccharide-protein conjugate) by methods known in the art, such as, for example, dialysis, concentration procedures, diafiltration procedures, tangential flow filtration, sedimentation, elution, centrifugation, sedimentation, ultrafiltration, depth filtration, and / or column chromatography (ion exchange chromatography, multimode ion exchange chromatography, DEAE, and hydrophobic interaction chromatography). Preferably, the polysaccharides are purified by a method comprising tangential flow filtration.

[0499] The purified polysaccharides can be activated (e.g., chemically activated) to render them capable of reacting (e.g., directly to a carrier protein or via a linker such as an eTEC spacer) and then incorporated into the glycoconjugates of the invention, as further described herein.

[0500] In one preferred embodiment, the saccharides of the invention are derived from the E. coli serotype 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.

[0501] As used herein, reference to any of the above serotypes refers to a serotype that includes a repeating unit structure (O unit, as described below) that is known in the art and unique to the corresponding serotype. For example, the term "O25a" serotype (also known in the art as serotype "O25") refers to a serotype that includes formula O25, as shown in Table 1. As another example, the term "O25b" serotype refers to a serotype that includes formula O25b, as shown in Table 1.

[0502] As used herein, serotypes are generally referred to herein unless otherwise specified, such as, for example, the term formula "O18" is generally referred to as including formula O18A, formula O18ac, formula O18A1, formula O18B, and formula O18B1.

[0503] As used herein, the term "O1" generally refers to encompassing species of formulas that include the generic designation "O1" in their formula names set forth in Table 1, such as any one of formula O1A, formula O1A1, formula O1B, and formula O1C, respectively, as set forth in Table 1. Thus, "O1 serotype" generally refers to serotypes that include any one of formula O1A, formula O1A1, formula O1B, and formula O1C.

[0504] As used herein, the term "O6" generally refers to species of formulae that include the generic designation "O6" in their formula names set forth in Table 1, such as any one of the formulae O6:K2;K13;K15; and O6:K54, respectively, shown in Table 1. Thus, "O6 serotype" generally refers to serotypes that encompass any one of the formulae O6:K2;K13;K15; and O6:K54.

[0505] Other examples of terms that refer generally to species of formulas that include a generic term in the formula name listed in Table 1 include "O4," "O5," "O18," and "O45."

[0506] As used herein, the term "O2" refers to formula O2 as shown in Table 1. The term "O2 O antigen" refers to a saccharide that encompasses formula O2 as shown in Table 1.

[0507] As used herein, references to O antigens from the above serotypes refer to the saccharide comprising the formula labeled with the corresponding serotype name. For example, the term "O25B O antigen" refers to the saccharide comprising the formula O25B shown in Table 1.

[0508] As another example, the term "O1 O antigen" refers generally to saccharides that include formulas that include the term "O1," such as formula O1A, formula O1A1, formula O1B, and formula O1C, each shown in Table 1.

[0509] As another example, the term "O6 O antigen" refers generally to saccharides that include formulas containing the term "O6," such as formula O6:K2; formula O6:K13; formula O6:K15, and formula O6:K54, each shown in Table 1.

[0510] O polysaccharide As used herein, the term "O polysaccharide" refers to any structure containing an O antigen, provided that the structure does not contain whole cells or lipid A. For example, in one embodiment, the O polysaccharide comprises lipopolysaccharide without attached lipid A. Steps for removing lipid A are known in the art and include, for example, heat treatment with the addition of acid. An exemplary process involves treatment with 1% acetic acid at 100°C for 90 minutes. This process is combined with a process for isolating the removed lipid A. An exemplary process for isolating lipid A includes ultracentrifugation.

[0511] In one embodiment, O polysaccharide refers to a structure consisting of an 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 the repeating units of an O antigen without the core saccharide. 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. In another preferred embodiment, O polysaccharide refers to a structure comprising an O antigen and a core saccharide. In another embodiment, O polysaccharide refers to a structure comprising an O antigen, a core saccharide, and a KDO portion.

[0512] Methods for purifying O polysaccharides, including the core oligosaccharide, from LPS are known in the art. For example, after purification of LPS, the purified LPS can be hydrolyzed by heating in 1% (v / v) acetic acid at 100°C for 90 minutes, followed by ultracentrifugation at 142,000 x g for 5 hours at 4°C. The supernatant containing the O polysaccharide is lyophilized and stored at 4°C. In certain embodiments, deletion of capsule synthesis genes is described to allow for simple purification of O polysaccharide.

[0513] 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 the use of hydrazine as an agent for O polysaccharide preparation. Preparation of LPS can be accomplished by methods known in the art.

[0514] In certain embodiments, O polysaccharides purified from wild-type, modified, or attenuated Gram-negative bacterial strains that express (but do not necessarily overexpress) a Wzz protein (e.g., wzzB) are provided for use in conjugate vaccines. In preferred embodiments, O polysaccharide chains are purified from Gram-negative bacterial strains that express (but do not necessarily overexpress) a wzz protein for use as a vaccine antigen in a conjugate or complexed vaccine.

[0515] In one embodiment, the O polysaccharide is 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, 49 fold, 50 fold, 51 fold, 52 fold, 53 fold, 54 fold, 55 fold, 56 fold, 57 fold, 58 fold, 59 fold, 60 fold, 61 fold, 62 fold, 63 fold, 64 fold, 65 fold, 66 fold, 67 fold, 68 fold, 69 fold, 70 fold, 71 fold, 72 fold, 73 fold, 74 fold, 75 fold, 76 fold, 77 fold, 78 fold, 79 fold, 80 fold, 81 fold, 82 fold, 83 fold, 84 fold, 85 fold, In a preferred embodiment, the O polysaccharide has a molecular weight that is increased by at least 1-fold and at most 5-fold compared to the corresponding wild-type O polysaccharide. In another embodiment, the O polysaccharide has a molecular weight that is at least two-fold and at most four-fold increased compared to the corresponding wild-type O polysaccharide. The increase in molecular weight of the O polysaccharide compared to the corresponding wild-type O polysaccharide is preferably associated with an increase in the number of O antigen repeating units. In one embodiment, the increase in molecular weight of the O polysaccharide is due to a wzz family protein.

[0516] In one embodiment, the O polysaccharide has 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, 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, The O polysaccharides of the present invention have a molecular weight increased by 0, 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 O polysaccharides of the present invention have a molecular weight increased by at least 1 kDa 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 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 12 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 15 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 18 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 21 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 22 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 30 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 1 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 5 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 12 kDa and at most 100 kDa, hi one embodiment, the molecular weight is increased by at least 15 kDa and at most 100 kDa.In one embodiment, the molecular weight is increased by at least 20 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 1 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 5 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 12 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 15 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 18 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 30 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 90 kDa. In one embodiment, the molecular weight is increased by at least 12 kDa and at most 85 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 70 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 60 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 50 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 49 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 48 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 47 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 46 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 45 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 44 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 43 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 42 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 41 kDa.Such an increase in molecular weight of the O polysaccharide compared to the corresponding wild-type O polysaccharide is preferably associated with an increase in the number of O antigen repeating units. In one embodiment, the increase in molecular weight of the O polysaccharide is due to a wzz family protein.

[0517] 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, 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 1 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, and many more than 100 repeat units. Preferably, the saccharide comprises an increase of 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.

[0518] O antigen O antigens are part of the lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria. O antigens are located on the cell surface and are variable cellular components. The variability of O antigens provides the basis for serotyping of Gram-negative bacteria. The current Escherichia coli (E. coli) serotyping scheme includes O polysaccharides 1 to 181.

[0519] O antigens comprise repeating oligosaccharide units (O units), whose wild-type structures typically contain 2-8 residues derived from a wide range of sugars. An exemplary O unit for an Escherichia coli (E. coli) O antigen is shown in Table 1. An exemplary O unit for a Klebsiella pneumoniae (K. pneumoniae) O antigen is shown in Table 1a.

[0520] In one embodiment, a saccharide of the invention may be a single oligosaccharide unit. In one embodiment, a saccharide of the invention is one repeating oligosaccharide unit of the relevant serotype. In such an embodiment, the saccharide may 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.

[0521] In one embodiment, the saccharide of the present invention may be an oligosaccharide. Oligosaccharides have a small number of repeating units (typically 5-15 repeating units) and are typically derived synthetically or by hydrolysis of a polysaccharide. In such an embodiment, the saccharide may 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.

[0522] Preferably, all saccharides of the present invention and in the immunogenic compositions of the present invention are polysaccharides. High molecular weight polysaccharides can induce a specific antibody immune response due to epitopes present on their antigenic surface. Isolation and purification of high molecular weight polysaccharides are preferably contemplated for use in the conjugates, compositions and methods of the present invention.

[0523] In some embodiments, the number of repeating O units in each individual O-antigen polymer (and thus 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 (from 4 to over 100 repeating units). The term "modal length" refers to the number of repeating O units. Gram-negative bacteria often have two different Wzz proteins that confer two different O Ag modal chain lengths, one long and one short. Expression (not necessarily overexpression) of a wzz family protein (e.g., wzzB) in Gram-negative bacteria can allow for manipulation of O-antigen length to shift or bias bacterial production of a particular length range of O-antigens and enhance the production of high yields of high molecular weight lipopolysaccharides. In one embodiment, "short" modal length as used herein refers to a small number of repeating O units, e.g., 1 to 20 repeating O units. In one embodiment, "long" modal length as used herein refers to a number of repeating O units greater than 20, up to a maximum of 40. In one embodiment, a "very long" mode length as used herein refers to greater than 40 repeating O units.

[0524] In one embodiment, the sugar produced has an increase of at least 10 repeating units, 15 repeating units, 20 repeating units, 25 repeating units, 30 repeating units, 35 repeating units, 40 repeating units, 45 repeating units, 50 repeating units, 55 repeating units, 60 repeating units, 65 repeating units, 70 repeating units, 75 repeating units, 80 repeating units, 85 repeating units, 90 repeating units, 95 repeating units, or 100 repeating units compared to the corresponding wild-type O polysaccharide.

[0525] In another embodiment, the saccharide of the invention has 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 12 , 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 repeat unit increases. Preferably, the saccharide comprises an increase of 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.

[0526] Methods for determining the number of repeating units in a saccharide are also known in the art. For example, the number of repeating units (or "n" in a formula) can be calculated by dividing the molecular weight of the polysaccharide (not including the molecular weight of 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 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 a formula is known in the art. For example, the molecular weight of the repeating unit of formula O25b is about 862 Da. For example, the molecular weight of the repeating unit of formula O1a is about 845 Da. For example, the molecular weight of the repeating unit of formula O2 is about 829 Da. For example, the molecular weight of the repeating unit of formula O6 is about 893 Da. When determining the number of repeating units in a conjugate, the molecular weight of the carrier protein and the protein:polysaccharide ratio are taken into consideration. As defined herein, "n" refers to the number of repeating units in a polysaccharide molecule (shown in parentheses in Table 1). As is known in the art, in biopolymers, repeat structures may incorporate regions of imperfect repeats, e.g., 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 such cases, n may represent the mean or median value for n of the molecules in the population.

[0527] In one embodiment, the O polysaccharide has an increase of at least one repeating unit of the O antigen 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 has an increase of at least one repeating unit of the O antigen 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 has an increase of at least one repeating unit of the O antigen 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 has an increase of at least one repeating unit of the O antigen compared to the corresponding wild-type O polysaccharide. 2, 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 at most 80 repeating units. In another embodiment, the O polysaccharide has 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23 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 repeat unit increases.In one embodiment, the saccharide comprises an O antigen where n in any of the O antigen formulas (such as, for example, those 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 can be combined with any maximum value to define a range. Exemplary ranges include, for example, at least 1 and at most 1000; at least 10 and at most 500; and at least 20 and at most 80, preferably at most 90. In one preferred embodiment, n is at least 31 and at most 90. In a preferred embodiment, n is 40 to 90, more preferably 60 to 85.

[0528] In one embodiment, the saccharide comprises an O antigen wherein n in any one of the O antigen formulas is at least 1 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 5 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 10 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 25 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 50 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 75 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 100 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 125 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 150 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 175 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 1 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 5 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 10 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 25 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 50 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 75 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 1 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 5 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 10 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 20 and at most 75.In one embodiment, n in any one of the O antigen formulas is at least 25 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 30 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 40 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 50 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 30 and at most 90. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 85. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 70. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 60. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 50. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 49. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 48. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 47. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 46. In one embodiment, n in any one of the O antigen formulas is at least 36 and at most 45. In one embodiment, n in any one of the O antigen formulas is at least 37 and at most 44. In one embodiment, n in any one of the O antigen formulas is at least 38 and at most 43. In one embodiment, n in any one of the O antigen formulas is at least 39 and at most 42. In one embodiment, n in any one of the O antigen formulas is at least 39 and at most 41.

[0529] For example, in one embodiment, n in the sugar is 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, or 90, most preferably 40. In another embodiment, n is at least 35 and at most 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 and 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.

[0530] Sugar structures can be determined by methods and means known in the art, such as, for example, NMR, including 1D, 1H, and / or 13C, 2D TOCSY, DQF-COSY, NOESY, and / or HMQC.

[0531] In some embodiments, the purified polysaccharide prior to conjugation has a molecular weight of between 5 kDa and 400 kDa. In other such embodiments, the saccharide is between 10 kDa and 400 kDa; 5 kDa and 400 kDa; 5 kDa and 300 kDa; 5 kDa and 200 kDa; 5 kDa and 150 kDa; 10 kDa and 100 kDa; 10 kDa and 75 kDa; 10 kDa and 60 kDa; 10 kDa and 40 kDa; 10 kDa and 100 kDa; 10 kDa. It has a molecular weight of a~200kDa; 15kDa~150kDa; 12kDa~120kDa; 12~75kDa; 12~50kDa; 12~60kDa; 35kDa~75kDa; 40kDa~60kDa; 35kDa~60kDa; 20kDa~60kDa; 12kDa~20kDa; or 20kDa~50kDa. In further embodiments, the polysaccharide has a molecular weight of 7 kDa to 15 kDa; 8 kDa to 16 kDa; 9 kDa to 25 kDa; 10 kDa to 100 kDa; 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 whole integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0532] 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).

[0533] Polysaccharides may become slightly reduced in size during normal purification procedures. Additionally, as described herein, polysaccharides can be subjected to sizing techniques prior to conjugation. Mechanical or chemical sizing can be used. Chemical hydrolysis can be performed using acetic acid. Mechanical sizing can be performed using high-pressure homogenization shear. The molecular weight ranges listed above refer to purified polysaccharides prior to conjugation (e.g., prior to activation).

[0534] Table 1-1

[0535] Table 1-2

[0536] Table 1-3

[0537] Table 1-4

[0538] Table 1-5

[0539] Table 1-6

[0540] Table 1-7

[0541] Table 1-8

[0542] Table 1-9

[0543] Table 1-10

[0544] Core oligosaccharides 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 the portion of the polysaccharide that contains the linkage between the O antigen and lipid A in wild-type E. coli. This linkage involves a ketosidic bond between the hemiketal function of the innermost 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) residue and the hydroxyl group of the GlcNAc residue of lipid A. The core oligosaccharide region shows high similarity among wild-type E. coli strains. It usually contains a limited number of sugars. The core oligosaccharide comprises an inner core region and an outer core region.

[0545] More specifically, the inner core is composed primarily of L-glycero-D-manno-heptose (heptose) and KDO residues. The inner core is highly conserved. The KDO residue has the following formula: KDO:

[0546] [ka] Includes.

[0547] The outer region of the core oligosaccharide shows greater variation than the inner core region, and differences in this region distinguish the five chemotypes in Escherichia coli (E. coli): R1, R2, R3, R4, and K-12. Hep II is the last residue of the inner core oligosaccharide. All outer core oligosaccharides share a structural theme, with a (hexose)3 carbohydrate backbone and two side chain residues, but the order of the hexoses in the backbone and the nature, position, and linkage of the side chain residues can all vary. The structures of the R1 and R4 outer core oligosaccharides are very similar, differing only in a single β-linked residue.

[0548] The core oligosaccharide of wild-type E. coli is classified in the art into five different chemotypes based on the structure of the distal oligosaccharide: E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12.

[0549] In a preferred embodiment, the compositions described herein comprise a glycoconjugate comprising a core oligosaccharide in which the O polysaccharide is bound to an O antigen. In one embodiment, the composition induces an immune response against at least one of the core E. coli chemotypes: 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 chemotypes. In another embodiment, the composition induces an immune response against at least three core E. coli chemotypes. In another embodiment, the composition induces an immune response against at least four core E. coli chemotypes. In another embodiment, the composition induces an immune response against all five core E. coli chemotypes.

[0550] In another preferred embodiment, the compositions described herein comprise a saccharide conjugate in which the O polysaccharide does not comprise a core oligosaccharide bound to an O antigen. In one embodiment, such a composition induces an immune response against at least 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 saccharide conjugate having the O polysaccharide does not comprise a core oligosaccharide.

[0551] E. coli serotypes can be characterized according to one of five chemotypes. Table 2 lists exemplary serotypes characterized according to chemotype. Bold serotypes represent serotypes most commonly associated with the indicated core chemotype. Thus, in preferred embodiments, the composition induces an immune response against at least one of the core E. coli chemotypes: E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12, including an immune response against any one of the corresponding E. coli serotypes.

[0552] [Table 2]

[0553] In some embodiments, a composition comprises a saccharide comprising a structure derived from a serotype having an R1 chemotype, e.g., selected from saccharides having formula O25a, formula O6, formula O2, formula O1, formula O75, formula O4, formula O16, formula O8, formula O18, formula O9, formula O13, formula O20, formula O21, formula O91, and formula O163, where n is 1 to 100. In some embodiments, the saccharide in the composition further comprises an E. coli R1 core moiety.

[0554] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R1 chemotype, e.g., selected from saccharides having formula O25a, O6, O2, O1, O75, O4, O16, O18, O13, O20, O21, O91, and O163, where n is 1-100, preferably 31-100, more preferably 35-90, and most preferably 35-65. In some embodiments, the saccharide in the composition further comprises an E. coli R1 core moiety in the saccharide.

[0555] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R2 chemotype, e.g., selected from saccharides having formula O21, O44, O11, O89, O162, and O9, where n is 1-100, preferably 31-100, more preferably 35-90, and most preferably 35-65. In some embodiments, the saccharide in the composition further comprises an E. coli R2 core moiety.

[0556] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R3 chemotype, e.g., selected from saccharides having formula O25b, O15, O153, O21, O17, O11, O159, O22, O86, and O93, where n is 1-100, preferably 31-100, more preferably 35-90, and most preferably 35-65. In some embodiments, the saccharide in the composition further comprises an E. coli R3 core moiety.

[0557] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R4 chemotype, e.g., selected from saccharides having formula O2, formula O1, formula O86, formula O7, formula O102, formula O160, and formula O166, where n is 1-100, preferably 31-100, more preferably 35-90, and most preferably 35-65. In some embodiments, the saccharide in the composition further comprises an E. coli R4 core moiety.

[0558] 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 the formula O25b and a saccharide having the formula O16, where n is 1-1000, preferably 31-100, more preferably 35-90, and most preferably 35-65). In some embodiments, the saccharide in the composition further comprises an E. coli K-12 core moiety.

[0559] In some embodiments, the saccharide comprises a core saccharide. 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.

[0560] In some embodiments, the saccharide does not comprise a core saccharide. 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.

[0561] Sugars and / or polypeptides derived from Klebsiella pneumoniae or fragments thereof Klebsiella pneumoniae is a Gram-negative pathogen known to cause urinary tract infections, bacteremia, and sepsis. In one aspect, any of the compositions disclosed herein may further comprise at least one saccharide that is or is derived from at least one K. 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 of the compositions disclosed herein may further comprise a polypeptide derived from K. pneumoniae selected from a polypeptide or immunogenic fragment thereof derived from a K. pneumoniae type I pilus protein; and a polypeptide or immunogenic fragment thereof derived from a K. pneumoniae type III pilus protein.

[0562] As is known in the art, K. pneumoniae O1 and O2 antigens contain homopolymeric galactose units (or galactans). K. 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 it has a D-galactan II cap structure. D-galactan III (d-Gal-III) is a variant of D-galactan I. In some embodiments, the saccharide derived from K. pneumoniae O1 comprises a repeating unit of [→3)-β-D-Galf-(1→3)-α-D-Galp-(1→]. In some embodiments, the saccharide derived from K. pneumoniae O1 comprises a repeating unit of [→3)-α-D-Galp-(1→3)-β-D-Galp-(1→]. In some embodiments, the saccharide derived from K. pneumoniae O1 comprises a repeating unit of [→3)-α-D-Galp-(1→3)-β-D-Galp-(1→]. Saccharides derived from Klebsiella pneumoniae O1 include repeating units of [→3)-β-D-Galf-(1→3)-α-D-Galp-(1→] and [→3)-α-D-Galp-(1→3)-β-D-Galp-(1→]. In some embodiments, saccharides derived from Klebsiella pneumoniae O1 include repeating units of →3)-β-D-Galf-(1→3)-[α-D-Galp-(1→4)]-α-D-Galp-(1→] (referred to as D-Gal-III repeating units).

[0563] In some embodiments, the saccharide derived from K. pneumoniae O2 comprises a repeating unit of [→3)-α-D-Galp-(1→3)-β-D-Galf-(1→] (which may be an element of the K. pneumoniae serovar O2a antigen). In some embodiments, the saccharide derived from K. pneumoniae O2 comprises a repeating unit of [→3)-β-D-GlcpNAc-(1→5)-β-D-Galf-(1→] (which may be an element of the K. pneumoniae serovar O2c antigen). In some embodiments, the saccharide derived from K. pneumoniae O2 comprises a modification of the O2a repeating unit by the side chain addition of a (1→4) linked Galp residue (which may be an element of the K. pneumoniae O2afg antigen). In some embodiments, the saccharide derived from K. pneumoniae O2 comprises a modification of the O2a repeating unit by the side chain addition of a (1→2) linked Galp residue (which may be an element of the K. pneumoniae O2aeh antigen).

[0564] Without being bound by mechanism or theory, it has been disclosed in the art that the O-antigen polysaccharide structures of Klebsiella pneumoniae serotypes O3 and O5 are identical to those of Escherichia coli (E. coli) serotypes O9a (formula O9a) and O8 (formula O8), respectively.

[0565] In some embodiments, the saccharide derived from K. pneumoniae O4 comprises a repeating unit of [→4)-α-D-Galp-(1→2)-β-D-Ribf-(1→)]. In some embodiments, the saccharide derived from K. pneumoniae O7 comprises repeating units of [→2-αL-Rhap-(1→2)-β-D-Ribf-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→]. In some embodiments, the saccharide derived from K. pneumoniae O8 serovar comprises the same repeating unit structure as K. pneumoniae O2a, but is non-stoichiometrically O-acetylated. In some embodiments, the saccharide derived from K. pneumoniae O12 serovar comprises repeating units of [α-Rhap-(1→3)-β-GlcpNAc] disaccharide repeating units.

[0566] [Table 3]

[0567] As used herein, the term "about" means within a statistically significant range of values, such as a stated concentration range, time frame, molecular weight, temperature, or pH. Such a range may 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, such a range may be within the experimental error typical of the standard method used to measure and / or determine a given value or range. The allowable variation encompassed by the term "about" depends on the particular system under test and is readily apparent to those skilled in the art. Whenever a range is described within this application, all numbers within that range are also contemplated as embodiments of the present disclosure.

[0568] It is intended by the inventors that the terms "comprising," "comprise," and "comprises" herein may be interchangeable in all instances with the terms "consisting essentially of," "consist essentially of," "consists essentially of," "consisting of," "consist of," and "consists of," respectively.

[0569] An "immunogenic amount," "immunologically effective amount," "therapeutically effective amount," "prophylactically effective amount," or "dose," each used interchangeably herein, generally refers to that amount of an antigen or immunogenic composition sufficient to elicit an immune response, either a cellular (T cell) or humoral (B cell or antibody) response, or both, as measured by standard assays known to those skilled in the art.

[0570] Any whole integer within any of the ranges in this document is contemplated as an embodiment of the present disclosure.

[0571] All references or patent applications cited within this patent specification are hereby incorporated by reference.

[0572] The present invention is illustrated in the accompanying examples. The following examples are carried out using standard techniques that are well known and routine to those skilled in the art, except where otherwise described in detail. The examples are illustrative but not limiting of the invention. [Example]

[0573] Example 1 Escherichia coli (E. coli) and Salmonella enterica (S. enterica) strains Clinical strains and derivatives are listed in Table 3. Additional reference strains included O25K5H1, a clinical O25a serotype strain; and Salmonella enterica serotype Typhimurium strain LT2.

[0574] A gene knockout in an E. coli strain was constructed that removes the target open reading frame but leaves a short scar sequence.

[0575] For simplicity, the hydrolyzed O antigen chains and core sugars are shown next to the O polysaccharide (OPS).

[0576] [Table 4]

[0577] Example 2 Oligonucleotide primers for cloning WZZB, FEPE, and O antigen gene clusters

[0578] [Table 5]

[0579] Example 3 Plasmid Plasmid vectors and subclones are listed in Table 5. PCR fragments carrying various E. coli and Salmonella wzzB and fepE genes were amplified from purified genomic DNA and subcloned into the high-copy-number plasmid provided in the Invitrogen PCR® Blunt cloning kit. This plasmid is based on the pUC replicon. Primers P3 and P4 were used to amplify the E. coli wzzB gene along with its native promoter, which are designed to bind to regions in the proximal and distal genes encoding UDP-glucose-6-dehydrogenase and phosphoribosyl adenine nucleotide hydrolase, respectively (annotated in Genbank MG1655 NC_000913.3). A PCR fragment containing the Salmonella fepE gene and promoter was amplified using previously described primers. Similar E. coli fepE primers were designed based on available Genbank genome sequences or internally generated whole-genome data (for GAR2401 and O25K5H1). The low-copy-number plasmid pBAD33 was used to express the O-antigen biosynthesis genes under the control of the arabinose promoter. The plasmid was first modified to facilitate cloning (by the Gibson method) of long PCR fragments amplified using universal primers homologous to the 5' promoter and 3' 6-phosphogluconate dehydrogenase (gnd) gene (Table 5).

[0580] [Table 6] Example 4 O antigen purification The fermentation broth was treated with acetic acid to a final concentration of 1-2% (final pH of 4.1). OAg extraction and delipidation were achieved by heating the acid-treated broth to 100°C for 2 hours. At the end of acid hydrolysis, the batch was cooled to ambient temperature, and 14% NH4OH was added to achieve a final pH of 6.1. The neutralized broth was centrifuged, and the centrate was collected. CaCl2 in sodium phosphate was added to the centrate, and the resulting slurry was incubated at room temperature for 30 minutes.

[0581] 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, which retained the 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 containing 2 M ammonium sulfate as the running buffer. OAg was collected in the effluent. For the long OAg, the HIC filtrate was concentrated and then buffer exchanged against water (20 dialysis volumes) using a 5 kDa membrane. For the short (native) OAg polysaccharide, the MWCO was further reduced to enhance yield.

[0582] In another embodiment, solids were removed by centrifugation, and the centrate was concentrated 12-fold 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, retaining OAg, was then purified using a carbon filter. The carbon filtrate was further purified by ion exchange (IEX) membrane chromatography. The IEX filtrate was then diluted 1:1 (v / v) with 4.0 M ammonium sulfate. The final ammonium sulfate concentration was 2 M. The ammonium sulfate-treated IEX filtrate was further purified using an HIC membrane containing 2 M ammonium sulfate as the running buffer. OAg was collected in the effluent. The HIC filtrate was concentrated and then buffer exchanged against water (20 dialysis volumes) using a 5 kDa membrane.

[0583] Background for Examples 5-17: The examples illustrated here demonstrate a platform-based process for the purification of O-antigen polysaccharides (also referred to as O-antigens or O-Ags) of all serotypes, which may contain inner / outer oligosaccharides.

[0584] The purification process described herein is applicable to both short- and long-chain O-Ag polysaccharides. Most examples given herein relate to long-chain O-Ags, except for Examples 10 and 11, which are short-chain O-Ags for E. coli serotypes O8 and O9, respectively.

[0585] Example 5 A method for purifying Escherichia coli (E. coli) O-antigen polysaccharides. 1. Release of O antigen This process begins with acid hydrolysis after fermentation to release O-Ag from lipopolysaccharide (LPS). This was achieved by treating a crude suspension of serotype O25b cell culture with acetic acid to a final concentration of 1.0% (v / v), bringing the pH to approximately 4.0. The acid broth was then heated to 100°C and incubated for 2.0 hours. After product release, the batch was cooled to ambient temperature of 20-30°C.

[0586] To further refine the release conditions of O25b O-Ag, a design of experiments (DOE) was set up to examine the effects of pH, temperature, and holding time on %KDO, concentration, molecular weight (MW), and O-acetate. The factors examined in the DOE study are listed in Table 1-1. Note that the initial concentration, upper limit of KDO, and upper limit of O-acetate were set at 3.5 mg / mL, 2%, and 1.0 mM, respectively, for this study.

[0587] [Table 7]

[0588] The predicted responses of concentration, %KDO, and O-acetate were evaluated at 1.0, 2.0, 2.5, and 4.0 h, respectively. The molecular weights for all conditions were uniformly approximately 50–54 kDa, except for the 80 °C temperature condition, where the MW was large. This was likely due to incomplete release of the product, in which case the O-Ag might be bound to cellular components or other nonspecific surface polysaccharides. Therefore, the DOE model was unable to provide a predicted response for MW at these conditions. At the 4.0 h time point, there was no %KDO within the range.

[0589] Based on the results of this DOE, 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. These release conditions were used for all other serotypes of O-Ag polysaccharide in subsequent experiments.

[0590] 2.Agglutination The primary purpose of this step is to precipitate cell debris, host cell proteins, and nucleic acids from the broth containing the released product. It also enhances the efficiency of downstream clarification unit operations. The acidified broth after product release from step 1 was treated with a 10% alum solution to a final concentration of 2% (w / v), and the pH was further adjusted to 3.2 using sulfuric acid. The flocculated slurry was incubated at ambient temperature for 1.0 h and then centrifuged at 12,000–14,000 g for 30 min. The supernatant was then filtered through a 0.2 μm filter or another suitable depth filter to remove small particles that may have drifted into solution. The depth filtrate was used for the initial purification of UFDF-1.

[0591] Alternatively, the acidified broth was neutralized to a pH of 6.0-7.0. The neutralized filtrate was centrifuged at 12,000 g for 30 minutes. The neutralized supernatant was 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 will go through the flocculation process described in the paragraph above. Subsequent purification steps exemplified in this example use this flocculation method unless otherwise noted.

[0592] A comparison of the SEC-HPLC chromatographic profiles for the neutralized filtrate containing the product and the depth filtrate after flocculation was performed. Both refractive index (RI) and UV280 detection showed that the flocculation step removed a substantial amount of impurities inherited from the fermentation medium.

[0593] 3. Ultrafiltration / Diafiltration (UFDF-1) The depth filtrate from step 2 above is further purified by ultrafiltration and diafiltration (UFDF) using a 10 kDa Sartocon Hydrosart membrane. The amount of depth filtrate processed is typically 1000 ml per m of membrane area. 2 The volume was 20-30 liters per 100 ml. The purpose of this operation was (i) volume reduction by concentrating the solution 10-20 times and (ii) buffer exchange by replacing the fermentation medium with the desired buffer via diafiltration. The buffer used in this step was 20 mM citrate / 0.1 M NaCl pH 6.0, followed by a second buffer of 20 mM Tris / 20 mM NaCl pH 7.2. The number of diafiltration volumes was 10 for both diafiltration steps. The retentate from UFDF was collected and analyzed. The conductivity and UV profiles during the UFDF run indicate that most low-molecular-weight and UV-related impurities were removed during the first diafiltration, as evidenced by a significant decrease in the UV signal for the permeate. Comparison of SEC-HPLC chromatograms of the depth filtrate and the retentate of UFDF-1 with respect to both RI and UV detection was analyzed.

[0594] 4. Carbon Filtration This unit operation reduces the levels of host cell impurities such as proteins and nucleic acids, as well as color impurities (see WO2008118752). 3M R32SP carbon filters were used, each with a carbon filter area of ​​1 m. 2 The carbon filter was first rinsed with water and then with diafiltration buffer at approximately 20 liters of buffer per square meter of filter area. The retentate from UFDF-1 was then filtered at 50 LMH (liters / m²) in single-pass mode. 2 The filter was then rinsed with buffer and the filtrate, including the rinse containing the product, was collected as the carbon filtrate.

[0595] SEC-HPLC chromatograms for the UFDF retentate and carbon filtrate show that RI and UV280 related impurities were removed and the carbon filtrate became visually colorless.

[0596] 5. IEX Membrane Chromatography This step was originally developed for the O antigens of serotypes O2 and O6 to remove nonspecific negatively charged impurities (see Examples 6 and 15). Thus, impurities derived 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.

[0597] The IEX membranes used here are NatriFlo membrane cassettes from Millipore. Alternatively, Sartobind Q membranes from Sartorius Stedim can be used. All examples exemplified here used NatriFlo membranes (hereinafter referred to as HD-Q) for IEX membrane chromatography unless otherwise indicated.

[0598] The membrane was first equilibrated with 20 mM Tris / 20 mM NaCl pH 7.2, typically 20–30 membrane volumes (MV). The carbon filtrate from the previous step was pumped through the membrane at a flow rate of 30–40 mL / min, along with approximately 200–250 mg of O-Ag per mL of MV. The product-containing flow-through or filtrate was collected. The membrane was rinsed with equilibration buffer and then washed with a high-salt buffer of 20 mM Tris / 1.0 M NaCl pH 7.2. The conductivity and UV280 profiles of the IEX membrane chromatography run from this profile showed a peak eluting during the high-salt wash, indicating the presence of an unknown negatively charged impurity in the carbon filtrate.

[0599] SEC-HPLC chromatograms for the carbon 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, suggesting that this unknown substance has a stronger ionic strength than O25b O-Ag.

[0600] 6. Hydrophobic Interaction Chromatography (HIC) This unit operation removes impurities with hydrophobic properties, such as residual lipid A, remaining from the acid hydr...

Claims

1. 1. A method for purifying bacterially derived sugars from a solution containing said sugars and contaminants after fermentation, comprising the steps of: a) Acid hydrolysis; b) agglomeration; c) First Ultrafiltration / Diafiltration-(UFDF-1); d) carbon filtration; e) chromatography; and f) Second Ultrafiltration / Diafiltration—(UFDF-2) wherein the agglomeration in step b) comprises adding a flocculating agent to the solution containing the sugar and contaminants, and the agglomeration in step b) is carried out at a temperature between 50°C and 70°C.

2. 2. The method of claim 1, wherein the chromatography in step e) comprises IEX membrane chromatography or hydrophobic interaction chromatography (HIC) or both.

3. 3. The method of claim 1 or 2, wherein the bacterium is a gram-positive bacterium.

4. 4. The method of claim 3, wherein the bacterium is any one of Streptococcus, Staphylococcus, Enterococci, Bacillus, Corynebacterium, Listeria, Erysipelothrix, or Clostridium.

5. The bacteria include Streptococcus pneumoniae, Streptococcus pyogenes, pyogenes), Streptococcus agalactiae or Group C and G Streptococci or Staphylococcus aureus 5. The method of claim 4, wherein the strain is any one of the strains selected from the group consisting of S. aureus, ...

6. 3. The method of claim 1 or 2, wherein the bacterium is a gram-negative bacterium.

7. 7. The method of claim 6, wherein the bacterium is any one of Haemophilus, Neisseria, Escherichia, or Klebsiella.

8. The bacteria include Haemophilus influenzae, Neisseria meningitidis, 8. The method of claim 7, wherein the bacterial strain is N. meningitidis, Escherichia coli, or Klebsiella pneumoniae.

9. Bacteria, 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, Formulas O14, O15, O16, O17, O18, O18A, O18ac, O18A1, O18B, O18B1, O19, O20, O21, O22, O23, O23A, O24, O25, O25a, O25b, O26, O27, O28 Formulas O29, O30, O32, O33, O34, O35, O36, O37, O38, O39, O40, O41, O42, O43, O44, O45, O45, O45rel, 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, O73, O73, O74, O75, O76, O77, Formulas O78, O79, O80, O81, O82, O83, O84, O85, O86, O87, O88, O89, O90, O91, O92, O93, O95, O96, O97, O98, O99, O100, O101, O102, O 103, 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, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O12 ...28, Formula O129, Formula O120, Formula O121, Formula O122, Formula O122, Formula O122, Formula O123, Formula O124, Formula O125, Formula O122, Formula O123, Formula O124, Formula O125, Formula O123, Formula O124, Formula O125, Formula O125, Formula O124, Formula O125, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O120, Formula O121, Formula O122, Formula O123, Formula O124, Formula O125, Formula O123, Formula O124, Formula O125, Formula O125, Formula O124, Formula O125, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O120, Formula O120, Formula O120, Formula O120, Formula O120, Formula O120, Formula O120, Formula O120, Formula O120, Formula O120, Formula O Equations O125, O126, O127, O128, O129, O130, O131, O132, O133, O134, O135, O136, O137, O138, O139, O140, O141, O142, O143, O144, and O145. Equations O146, O147, O148, O149, O150, O151, O152, O153, O154, O155, O156, O157, O158, O159, O160, O161, O162, O163, O164, O165, O166a saccharide having a structure selected from any one of 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; 9. The method of claim 8, wherein the vector is E. coli.

10. The present invention relates to a method for treating a bacterial infection, the method comprising the steps of: (a) administering to a patient a saccharide having a structure selected from any one of formulas K.O1.1, K.O1.2, K.O1.3, K.O1.4, K.O2.1, K.O2.2, K.O2.3, K.O2.4, K.O3, K.O4, K.O5, K.O7, K.O12, or K.O8; 9. The method of claim 8, wherein the bacterium is Bacillus subtilis (Bacillus subtilis).

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