Low contamination antimicrobial vaccine

Abstract

A vaccine composition comprising an oligosaccharide β - (1 → 6) - glucosamine antigen attached via a linker to a tetanus toxoid monomer carrier for providing immunity against a microorganism having a cell wall structure comprising polymeric N-acetyl - β - (1 → 6) - glucosamine structures wherein up to about 20 percent of the N-acetyl groups are deacetylated (PNAG structures), wherein the vaccine composition does not induce denaturation of the tetanus toxoid into oligomers.SOLUTION: There is provided a vaccine composition comprising a vaccine in which an antigen consisting of a penta - β - (1 → 6) - glucosamine group represented by the following partial structural formula is attached to a tetanus toxoid monomer carrier via a linker.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 934,925, filed November 13, 2019, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to compositions containing oligosaccharide β-(1→6)-glucosamine groups. These vaccine compositions provide immunity against microorganisms with cell wall structures containing polymeric N-acetyl-β-(1→6)-glucosamine structures ("PNAG structures") in which up to about 20 percent of the N-acetyl groups in the polymer are deacetylated. [Background technology]

[0003] Vaccines containing oligosaccharide β-(1→6)-glucosamine antigens attached via a linker to a toxoid carrier are known. These vaccines generate cytotoxic antibodies in vivo against microorganisms containing PNAG structures in their cell walls. The antibodies thus generated combine with complement and other components of the immune system to kill these microorganisms.

[0004] A vaccine using tetanus toxoid as a carrier to which multiple copies of oligosaccharides are attached is disclosed in U.S. Serial No. 10 / 713,790, which is incorporated herein by reference in its entirety. Traditionally, attachment of the oligosaccharide group to the toxoid is accomplished via a covalent linker to a reactive amino group on the toxoid (e.g., -NH2 found on lysine residues). While the chemistry is well established, there are many complexities in working with toxoid chemistry.

[0005] First, tetanus toxoid is prepared by treating tetanus toxin with chemicals such as formaldehyde, which renders it nontoxic but still antigenic. Formaldehyde reacts with the toxin's reactive amino groups. Furthermore, amino acids such as glycine and lysine are added to stabilize the toxoid and prevent it from reverting to a toxin. Second, in addition to unreacted amounts of glycine and lysine, the manufacturing process also releases fragments of the toxin / toxoid into the toxoid composition. These fragments contain one or more amino groups.

[0006] The tetanus toxoid is then reacted with a spacer arm that has two functional groups—one that bonds with a reactive amino group on the toxoid, and a second that is orthogonal to the first and reacts with a complementary functional group on the aglycone. Furthermore, the spacer arm increases the distance between the added oligosaccharide and the toxoid. The oligosaccharide aglycone then combines with the spacer arm-bearing tetanus toxoid to form the vaccine compound. This is shown in the following reaction scheme:

[0007] [ka] where n represents the number of moles of bifunctional spacer arm, p represents the number of spacer arms-Y up to n added to the toxoid, and q represents the number of linker oligosaccharide groups combined with a given tetanus toxoid, with the proviso that q cannot be greater than p. Note that the aglycone attached to the oligosaccharide and the spacer arm attached to the toxoid combine to form the linker.

[0008] One problem with the above reaction arises from the presence of these amino groups containing contaminants in the toxoid composition during coupling of the oligosaccharide aglycone to the toxoid. Specifically, these amino groups can also react with the first reactive functional group of the spacer arm, which can then cause the second reactive functional group of the spacer arm to react with the complementary functional group of the aglycone, resulting in both the loss of the oligosaccharide aglycone and the production of undesirable impurities in the vaccine composition. This is illustrated in the following reaction:

[0009] [ka] where m is the total amino group content, m' is the proportion of reacted amino groups that is less than the total amino content m; and m'' is the proportion of reacted oligosaccharides that are bound to the contaminant that is less than m'.

[0010] These oligosaccharide-linker-NH contaminants are undesirable, especially those with molecular weights below about 100,000.

[0011] Additionally, tetanus toxoid has a tendency to oligomerize, such that the toxoid can exist in monomeric, dimeric, trimeric, and higher oligomeric forms (e.g., 4–10 toxoid units). As oligomerization progresses, the number of oligosaccharides that can be attached to the toxoid per monomer decreases, as does the surface area available per monomer for oligomerization. Therefore, oligomers of the toxoid, such as trimers and higher, are less desirable. While laborious purification processes can provide monomeric tetanus toxoid, these processes are complicated by the fact that the monomers tend to re-oligomerize over time. Summary of the Invention

[0012] In one embodiment, the present invention provides a vaccine composition comprising a pharmaceutically acceptable excipient and an effective amount of a vaccine comprising at least 10, preferably about 10 to about 40, oligomeric β-(1→6)-glucosamine group units linked via a linker to a tetanus toxoid carrier, wherein the oligomer comprises 3 to 12 repeating β-(1→6)-glucosamine units, with the proviso that less than about 40-some percent of the total number of such units are N-acetylated; the vaccine composition contains less than 3 percent of detectable impurities each having a molecular weight of less than 100,000; Additionally, the composition comprises monomeric and dimeric toxoids with less than 10 percent detectable higher order oligomers; Still further, the composition provides a vaccine composition that is maintained at a temperature sufficient to inhibit oligomerization of the toxoid without inducing denaturation.

[0013] In one embodiment, the present invention provides a vaccine composition comprising a pharmaceutically acceptable excipient and an effective amount of a vaccine comprising at least 25, and preferably about 30 to about 40, oligomeric β-(1→6)-glucosamine group units linked via a linker to a tetanus toxoid carrier, wherein the oligosaccharide group comprises 3 to 12 repeating β-(1→6)-glucosamine units, with the proviso that less than about 40-some percent of the total number of such units are N-acetylated; the vaccine composition contains less than 3 percent by weight of detectable impurities having a molecular weight of less than 50,000; Additionally, the composition comprises monomeric and dimeric toxoids with less than 5 percent detectable higher order oligomers; Still further, the composition provides a vaccine composition that is maintained at a temperature sufficient to inhibit oligomerization of the toxoid without inducing denaturation.

[0014] In one embodiment, the present invention provides a compound comprising a pharmaceutically acceptable excipient and an effective amount of a compound of Formula I: (AB) x -CI wherein A is a compound represented by the following formula:

[0015] [ka] or mixtures thereof, wherein B is a repeating β-(1→6)-glucosamine unit having the formula:

[0016] [ka] is of; The left side of the equation is attached to C, and the right side is attached to A; C is tetanus toxoid; x is an integer from about 10 to about 40; y is an integer between 1 and 10; R is hydrogen or acetyl, provided that no more than 40% of the R groups are acetyl. 1. A vaccine composition comprising a vaccine compound of the composition contains less than 3 percent of detectable impurities having a molecular weight of less than about 100,000; Additionally, the composition comprises monomeric and dimeric toxoids with less than about 5 percent detectable higher order oligomers; Still further, the composition relates to a vaccine composition that is maintained at a temperature sufficient to inhibit oligomerization of the toxoid without inducing denaturation.

[0017] In one embodiment of the above vaccine composition, the vaccine compound used therein is represented by formula II: (A'-B) x -C II wherein A' is of the following formula:

[0018] [ka] where B, C, and x are as defined above.

[0019] In one embodiment, the vaccine of the present invention provides effective immunity to a patient from microorganisms that contain polymeric N-acetyl-β-(1→6)-glucosamine groups in their cell walls, in which up to 20 percent of the N-acetyl groups of the polymer are deacetylated.

[0020] In one embodiment, the present invention provides a method for providing a patient with effective immunity from a microorganism that contains polymeric N-acetyl-β-(1→6)-glucosamine groups in its cell wall, wherein up to 20 percent of the N-acetyl groups of the polymer are deacetylated, comprising administering to the patient a pharmaceutical composition described herein.

[0021] Representative vaccine compounds of the present invention are shown in the table below.

[0022] [ka]

[0023] [Table 1]

[0024] In one embodiment, the compositions of the invention comprise no more than about 0.5 weight percent of oligosaccharide-bound contaminants having a particle size of less than 1 micron, said weight percent being based on the weight of the vaccine compound. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 shows the 1H NMR of compound 17 (described below). [Figure 2] FIG. 2 shows the 13C NMR of compound 17. [Figure 3] FIG. 3 provides an HPLC trace of the conversion of the disulfide, compound 16, to two equivalents of the monosulfide, compound 17. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention provides pharmaceutical compositions comprising oligosaccharide β-(1→6)-glucosamine groups.

[0027] The vaccine compositions described herein provide effective immunity to a patient against microbial infection, wherein the microorganism contains oligomeric N-acetyl-β-(1→6)-glucosamine structures in its cell wall, and up to 20 percent of the N-acetyl groups of the polymer are deacetylated.

[0028] Before describing the present invention in more detail, the following terms will first be defined. If a term used herein is not defined, it has its generally accepted scientific or medical meaning.

[0029] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0030] "Optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0031] When the term "about" is used before a numerical designation, for example, temperature, time, amount, concentration, and others including ranges, it indicates an approximation that may vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. Preferably, the term "about" when used in reference to a dose means that the dose may vary by + / - 10%.

[0032] "Comprising" or "comprises" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. When used to define compositions and methods, "consisting essentially of" shall mean excluding other elements that are essential to the combination for the stated purpose. Thus, a composition consisting essentially of the elements defined herein will not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. "Consisting of" shall mean excluding other components beyond trace elements and substantial method steps. Embodiments defined by each of these transitional phrases are within the scope of the present invention.

[0033] As used herein, "percent of detectable impurities" refers to area percent. This does not include higher oligomers. An assay for assessing area percent is described in Example 6 below. Impurities are assessed, for example, using size exclusion chromatography analysis as a percentage of the total area attributable to impurities. The referenced impurities can be separated and controlled by size exclusion chromatography and other molecular weight cutoff filtration processes. Buffer salts, pH, and process conditions can be used to ensure desired properties, quality, and stability.

[0034] As used herein, "inhibiting oligomerization of the toxoid in the vaccine without inducing denaturation" refers, in part, to operational parameters for manipulating a monomeric and / or dimeric toxoid vaccine. For example, monomeric and / or dimeric toxoids may exhibit long-term stability against oligomerization when the vaccine compositions disclosed herein are stored at temperatures ranging from about 2°C to about 8°C. Denaturation of the vaccine compositions disclosed herein may, in some embodiments, occur when the compositions are stored at or below 0°C. Similarly, oligomerization of monomeric and / or dimeric toxoids may occur when the vaccine compositions are stored at temperatures above 8°C, or more likely when stored at temperatures above 20°C.

[0035] As used herein, "detectable impurities" refers to low molecular weight impurities (molecular weight less than 100,000) resulting from toxoid degradation and can be measured by chromatographic separation using a detector. In embodiments, chromatographic separation can be by filtration, size exclusion chromatography, etc. Detectors can use UV detection means, refractive index, etc. Similarly, "percent detectable higher order oligomers" can be determined in a similar manner by chromatographic techniques coupled with detection.

[0036] As used herein, "oligosaccharide-linked and amino-containing contaminants" refer to two forms of contaminants that can form due to the presence of trace amounts of low-molecular-weight impurities. Oligosaccharide-linked contaminants include adducts formed between degradation impurities present in a toxoid preparation and the linking reagents used to attach oligosaccharides to the toxoid. Thus, oligosaccharide-linked contaminants include amine-containing degradation products from a toxoid preparation that are linked to the spacer arm that constitutes the linker via one or more amino groups of the degradation product, and to oligosaccharides attached distally to the spacer arm at amino groups of the contaminant. These by-product adducts consume reagents intended to react with the monomeric and / or dimeric toxoids, thus depleting the reagent supply. Reagent shortages can result in a second form of contaminant, namely, "amino-containing contaminants," which are unreacted amino groups present on the toxoid in the final vaccine composition. Embodiments herein are provided that minimize these products using the stepwise filtration methods described herein.

[0037] The term "β-(1→6)-glucosamine unit" or "glucosamine unit" refers to an individual glucosamine structure such as:

[0038] [ka] (where the 6-hydroxyl group is condensed with the 1-hydroxyl group of the preceding glucosamine unit, and the dashed lines represent the attachment sites to the preceding and following glucosamine units.) When combined with another "β-(1→6)-glucosamine unit," the resulting disaccharide structure is as follows:

[0039] [ka]

[0040] The term "β-(1→6)-glucosamine unit with an N-acetyl group" refers to the following structure:

[0041] [ka] (where the 6-hydroxyl group of the second unit is condensed with the 1-hydroxyl group of the preceding glucosamine unit).

[0042] The term "oligosaccharides containing β-(1→6)-glucosamine groups" refers to glucosamine groups on vaccine compounds that mimic portions of the cell wall of pathogenic bacteria; these are defined as "oligosaccharide β-(1→6)-glucosamine structures" (defined below). Again, such groups are limited to 3-12 β-(1→6)-glucosamine units, with up to 40% of the units having an N-acetyl group. In one embodiment, fewer than 30% of the β-(1→6)-glucosamine units are N-acetylated. In another embodiment, fewer than 20% of the β-(1→6)-glucosamine units are N-acetylated. In yet another embodiment, fewer than 10% of the β-(1→6)-glucosamine units are N-acetylated. In yet another embodiment, none of the β-(1→6)-glucosamine units are N-acetylated.

[0043] The terms "oligosaccharides containing N-acetyl β-(1→6)-glucosamine structures" or "polysaccharides containing N-acetyl β-(1→6)-glucosamine structures" refer to those structures found in microbial cell walls, in which up to 20 percent of the N-acetyl groups of the polymer are deacetylated. Microbial walls contain many of these structures, which are conserved across many microbial lineages. These structures are primarily N-acetyl β-(1→6)-glucosamine, but contain regions of deacetylated sugars due to the action of enzymes such as poly-beta-1,6-D-glucosamine-N-deacetylase. Thus, the vaccines of the present invention generate antibodies, including those that target such deacetylated oligosaccharide regions. Without being limited to any theory, antibodies against such deacetylated sugars are cytotoxic in vivo against such microorganisms.

[0044] As used herein, the terms "vaccine composition" and "pharmaceutical composition" refer to pharmaceutical compositions containing the compounds of Formulas I and II above, including an adjuvant and a pharmaceutical carrier. These compositions also contain limited amounts of oligosaccharide-linked and amino-containing contaminants, including those containing no more than 3 percent, preferably less than 2 percent, and more preferably less than 1 percent of such contaminants. These compositions provide effective immunity against any microorganism containing an oligosaccharide / polysaccharide with an N-acetyl-β-(1→6)-glucosamine structure in its cell wall. Therefore, unlike classical vaccines that vaccinate against a single bacterium, the vaccine compositions described herein can provide effective immunity against any microorganism containing the oligosaccharide structures described herein. Such microorganisms include, but are not limited to, Gram-positive bacteria, Gram-negative bacteria, antibiotic-resistant bacteria (e.g., methicillin-resistant Staphylococcus aureus), fungi, and the like.

[0045] As used herein, the term "effective immunity" refers to the ability of a defined amount of a vaccine composition to generate an antibody response in vivo sufficient to treat, prevent, or ameliorate a microbial infection, wherein the microorganism contains N-acetyl-β-(1→6)-glucosamine-containing oligosaccharides / polysaccharides in its cell wall.

[0046] Vaccine compounds refer to compounds of formula I and II. These compounds can exist as solvates, especially hydrates. Hydrates may form during the preparation of compounds or compositions containing compounds, or hydrates may form over time due to the hygroscopicity of compounds. Compounds of the present invention can also exist as organic solvent solvates, including DMF, ether, and alcohol solvates, among others. Identifying and preparing specific solvates is within the skill of those skilled in the art of synthetic organic or medicinal chemistry.

[0047] The term "toxoid" refers to monomeric and oligomeric forms of tetanus toxoid. Because oligomerization reduces the surface area of ​​each monomeric toxoid in the oligomer, the presence of oligomeric tetanus toxoid components reduces the average number of exposed reactive amino groups. This, in turn, results in a lower rate of oligosaccharide attachment to the toxoid. The vaccine compositions disclosed herein include toxoids in monomeric and / or dimeric form. In embodiments, the ratio of monomer to dimer ranges from about 10:1 to about 1:10, or from about 5:1 to about 1:5, or from about 2:1 to about 1:2.

[0048] "Subject" refers to a mammal. The mammal can be a human or non-human mammal, but is preferably a human.

[0049] "Treating" or "treatment" of a disease or disorder in a subject refers to 1) preventing the disease or disorder from occurring in a subject who is predisposed to or who does not yet exhibit symptoms of the disease or disorder; 2) inhibiting or arresting the development of the disease or disorder; or 3) ameliorating or causing regression of the disease or disorder.

[0050] An "effective amount" refers to an amount of a vaccine composition of the present invention sufficient to treat a disease or disorder afflicting a subject or to prevent such disease or disorder from occurring in said subject or patient.

[0051] "Reactive amino functionality" refers to the primary amino groups (-NH2) found in the lysine and guanidine side chains of tetanus toxoid, but does not include the amide (-NHC(O)-) groups found in the peptide bonds or amide side chains of tetanus toxoid, such as those found in glutamine.

[0052] "Low molecular weight amino compounds" refers to amino-containing compounds present as contaminants in tetanus toxoid compositions, including fragments of the toxoid, buffers containing amino groups, reaction quenchers such as lysine, glycine, and ammonium sulfate, toxin antidotes such as formalin, and other amino-containing reagents that come into contact with tetanus toxoid. Typically, such low molecular weight reactive amino compounds have a molecular weight of less than about 100,000, preferably less than 10,000.

[0053] General synthesis method The compounds of the present invention can be prepared from readily available starting materials using the following general methods and procedures. Where typical or preferred process conditions (i.e., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.) are given, it will be understood that other process conditions can also be used unless otherwise specified. Optimum reaction conditions may vary with the particular reactants or solvents used, although such conditions can be determined by one skilled in the art by routine optimization procedures.

[0054] Furthermore, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions.Proper protecting groups for various functional groups, as well as suitable conditions for protecting and deprotecting specific functional groups, are well known in the art.For example, many protecting groups are described in TW Greene and PGM Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and the references cited therein.

[0055] The starting materials for the following reactions are generally known compounds or can be prepared by known procedures or obvious modifications thereof. For example, many of the starting materials are available from commercial suppliers such as SigmaAldrich (St. Louis, Missouri, USA), Bachem (Torrance, California, USA), and Emka-Chemce (St. Louis, Missouri, USA). Others are described in Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wiley and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5, and Supplementals (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), and March's Advanced Organic Chemistry, (John Wiley and Sons, 5 th Organic synthesis compounds may also be prepared by procedures described in standard reference texts such as The American Journal of Biological Chemistry (Eds. Edition, 2001), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), or obvious modifications thereof.

[0056] Synthesis of Representative Vaccine Compounds of the Invention The general synthesis of the vaccine compounds of the present invention is known in the art and is disclosed in U.S. Patent Application No. 10 / 713,790 and U.S. Patent Nos. 7,786,255 and 8,492,364, each of which is incorporated herein by reference in its entirety.

[0057] In one embodiment of the vaccine compounds described herein, the β-(1→6)-glucosamine groups are limited to 4-6 units, and preferably 5 units, eg, in Formula I, y=2-4.

[0058] In some embodiments, compounds are homogeneous in that y is a single integer selected from 1 to 10, inclusive. Thus, compounds disclosed herein can be designed to be homogeneous with y=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, compounds of Formula I can be designed to be heterogeneous with two or more values ​​of y, such as a mixture of y=1 and 2, or y=2 and y=3, or y=3 and y=4, or y=4 and y=5, or y=5 and y=6, or y=6 and y=7, or y=7 and y=8, or y=8 and y=9, or y=9 and y=10. Such pairings of y need not be consecutive. Thus, compounds may include a mixture of any combination of two or more different values ​​for y, such as y=1 and y=3, or y=1 and y=4, or y=2 and y=4, or y=2 and y=5. In some embodiments, the compounds can be heterogeneous with 3 or more values ​​for y, or 4 or more values ​​for y, or 5 or more values ​​for y, all up to 10 different values ​​for y. In some embodiments, each occurrence of y is independent in the compound of Formula I.

[0059] In some embodiments, two or more compounds of Formula I can be used in pharmaceutical compositions in which each individual compound of Formula I is homogeneous in y, while other compounds of Formula I have different y values. In such embodiments, the homogeneous compounds used are simply mixed together in defined weight percentages. For example, a pharmaceutical composition can include a compound of Formula I where y=1 in a mixture with a compound of Formula I where y=2. When a pharmaceutical composition or method includes a heterogeneous mixture of compounds of Formula I, the mixture can be defined in terms of the relative weight percentages of each compound of Formula I. For example, a mixture can include 50 weight percent of a compound of Formula I where y=1 and 50 weight percent of a compound of Formula I where y=2. Any combination of compounds totaling 100% is contemplated; for example, one, two, three, four, five, or more compounds, each with a different y value, can be mixed in known relative weight percentages totaling 100%. Thus, any combination of weight percentages of compounds of Formula I can be used in the pharmaceutical compositions and methods disclosed herein. Thus, for combinations of two compounds of Formula I, the percentage can be expressed as the ratio of the two compounds and can be any range from 0.1:99.9 to 99.9:0.1, inclusive, and any value between, for example, 1:99, 5:95, 10:90, 15:85, 20:80, etc. up to 99:1 (including fractional values). Similarly, when three, four, five, or more compounds of Formula I are used in a pharmaceutical composition, the relative weight percentage of each compound can vary from 0.1 weight percent up to 99 weight percent, provided that the total amount of the different compounds of Formula I adds up to 100%.

[0060] Formation of the linker group is accomplished by art-recognized synthetic techniques, exemplified, but not limited to, those found in U.S. Patent No. 8,492,364 and the Examples below. In one embodiment, the first portion of the aglycone is attached to a reducing β-(1→6)-glucosamine unit and has the following formula III:

[0061] [ka] III (wherein y is an integer from 1 to 10; up to 40% of the amino groups may be N-acetyl groups). As shown in Figure 1, it carries a thiol (-SH) group.

[0062] Preparation of tetanus toxoid Tetanus toxoid is commercially available in various purity levels, and the toxoid always contains significant amounts of amino-derived contaminants, including fragments of the toxoid released by enzymatic or hydrolytic processes that contain amino functional groups, and amino-derived contaminants from unreacted glycine and arginine added during the conversion of the toxin to the toxoid.

[0063] In addition to low-molecular-weight contaminants, initial toxoid preparations may contain varying amounts of toxoid monomers, dimers, trimers, and higher-order oligomers. Oligomers containing three or more monomer units, when present in a toxoid preparation, impair the ability to produce high oligosaccharide loadings. Loading is the number of oligosaccharide units attached to a given monomeric or dimeric toxoid unit. Surprisingly, it has been discovered that the composition of tetanus toxoid monomers and dimers allows for suitable loadings, obviating the need to separate the monomeric toxoid from the dimeric toxoid. However, higher-order oligomers should be removed; in embodiments, higher-order oligomers should constitute less than 5% of detectable higher-order oligomers. Higher-order oligomers and low-molecular-weight contaminants can be removed by stepwise (or sequential) filtration while still providing a product with acceptable loading. In embodiments, the purified toxoid preparations disclosed herein have a loading of at least 25, preferably at least 30. Lower loadings are often the result of impurities reacting with the oligosaccharide conjugation chemistry, thereby reducing loading based on the stoichiometry of using a 35-fold excess of conjugation reagent relative to the theoretically available sites for reaction on the monomer and dimer toxoids. Note that the problem of the presence of impurities is not simply solved by using a large excess of reagent, as this is not economical and can cause purification problems due to nonspecific binding of the oligosaccharide reagent to the toxoid.

[0064] In embodiments, there is provided a method of using stepwise filtration to minimize both high molecular weight impurities, including higher oligomers of the toxoid, and low molecular weight impurities, including toxoid degradation products. In embodiments, the stepwise filtration method comprises filtration through one or more 3-5 micron pore size filters. These filters trap higher-order oligomers while allowing toxoid monomers and dimers to pass through. In embodiments, such filtration can be performed in stages, in that a first filtration can be performed using, for example, a 5 micron filter, followed by filtration through a 4 micron filter, and then a 3 micron filter. At 3 microns and above, it is expected that the majority of toxoid dimers and monomers will pass through the filter, while the filter material will trap higher-order oligomers. Without wishing to be bound by theory, it is hypothesized that filters with pore sizes of 3 microns and above will allow some toxoid monomers or dimers to pass through, since monomers are characterized as being approximately 2.5 microns in length and 0.5 microns in width.

[0065] In another filtration step, low molecular weight impurities can be removed by using a filter with a pore size of 2.5 microns or less. In embodiments, low molecular weight impurities are removed by passing the toxoid mixture through, for example, a filter with a pore size of 2.5 microns, with the monomeric and dimeric forms of the toxoid remaining on the filter and the low molecular weight impurities passing through the filter. In embodiments, the second step to remove low molecular weight impurities involves the use of a 2 micron filter, or in other embodiments, a 1.5 micron filter. In embodiments, the pore size used to remove low molecular weight impurities can be stepwise, such as decreasing the pore size from 2.5 microns to 1.5 microns.

[0066] In embodiments, both filtration steps to remove high and low molecular weight impurities can be performed before any functionalization chemistry to generate the oligosaccharide-toxoid covalent adduct is performed. Thus, in embodiments, a method for making a vaccine composition disclosed herein includes passing a toxoid preparation through a first filter to remove higher-order oligomeric impurities, with dimeric and monomeric toxoids passing through the first filter, and then, after passing through the first filter, passing the toxoid preparation through a second filter to remove low molecular weight impurities, with the monomeric and dimeric toxoids being retained on the filter and the smaller molecular weight impurities passing through the filter. In embodiments, after using both filters, the monomeric and dimeric toxoid mixture is reacted with a spacer arm to generate a linker to which an oligosaccharide can be covalently attached. Filters with appropriate pore sizes are known in the art and are commercially available from Spherotech, Inc., Lake Forest, Illinois, USA, www.spherotech.com / contact.htm.

[0067] In embodiments, low molecular weight impurities may be removed first by using smaller filter pore sizes, followed by removal of higher molecular weight impurities by larger filter sizes.

[0068] In embodiments, the reaction chemistry for attaching oligosaccharides can be performed between any filtration steps. For example, in embodiments, a first filtration can be performed to remove small impurities, and the higher-order oligomers, along with the monomers and dimers, can be reacted with a spacer arm, followed by the attachment of oligosaccharides. The adduct can then be subjected to a second filtration step to remove the higher-order oligomers. Similarly, only high molecular weight impurities can be removed, followed by the formation of oligosaccharide adducts, which can then be purified by a second filtration step of low molecular weight impurities. However, those skilled in the art will understand that removing low and high molecular weight impurities prior to the oligosaccharide attachment chemistry, if desired, maximizes the efficiency of the reagent reaction with the amino groups of the monomer and / or dimer toxoids.

[0069] As those skilled in the art will further appreciate, the oligosaccharide attachment described herein is performed in two steps, with stepwise filtration occurring before and / or after the first step of attaching the spacer arm. Thus, the spacer arm can be attached after either the high-molecular-weight filtration, the low-molecular-weight filtration, or both. Thus, the final oligosaccharide attachment chemistry may follow an intervening filtration step. The amino group of the contaminant first reacts with the spacer, as described above, to form an intermediate that reacts with the aglycone, as described above, to form the amino-derived contaminant containing oligosaccharide. These contaminants can account for the residue of the vaccine composition, typically in amounts ranging up to 20 weight percent based on the weight of the toxoid.

[0070] The second portion of the linker, as shown in Formula IV, is attached to the tetanus toxoid in the following manner:

[0071] [ka] IV

[0072] In this formula, the separate moieties of tetanus toxoid are shown with wavy lines and are merely illustrative in nature and are not intended to provide the complete structure of the toxoid. The disulfide bridge is represented by a single line connecting the moieties. For clarity, only a single second moiety of the linker is shown, although there are multiple such second moieties covalently attached to amino groups found on the toxoid.

[0073] When the first and second portions of the linker are combined under coupling conditions, a thioether bond is formed. The reaction is carried out in an inert diluent, optionally in the presence of a base, to remove the acid generated. The thioether bond connects the first and second portions of the linker, thereby providing a covalent bond to the oligosaccharide β-(1→6)-glucosamine group of the tetanus toxoid via the combination linker, as shown below for a vaccine compound where y is defined herein:

[0074] [ka] In the formula, up to 40% of the amino groups may be N-acetyl groups.

[0075] It is understood that the number of β-(1→6)-glucosamine group-linker groups attached to tetanus toxoid is stoichiometrically controlled so that from about 31 to about 39 of such groups are attached to the toxoid, thereby providing the vaccine compound of the present invention.

[0076] Methods, Uses and Pharmaceutical Compositions The vaccine composition of the present invention can initiate an effective immune response against microorganisms that have PNAG oligosaccharide β-(1→6)-glucosamine structures in their cell walls. An effective immune response develops approximately four weeks after inoculation into a patient. After an effective immune response is developed, the patient is provided with protection against subsequent microbial infections in which the microorganism in question has a cell wall that contains PNAG.

[0077] When used in this manner, the vaccine composition of the present invention is administered to patients at risk of microbial infection resulting from such microorganisms. Such patients include, by way of example only, the elderly, patients undergoing elective surgery, and patients traveling to destinations where microbial infections are prevalent. Vaccines are typically administered intramuscularly to immunocompetent patients along with a suitable adjuvant to enhance the immune response. After an incubation period, patients acquire natural immunity against such microorganisms. Such immunocompetent patients have an effective immune system capable of generating an immune response to the antigen. Preferably, such patients have an activated white blood cell (WBC) count of at least about 1,000 WBC per microliter, preferably at least about 1,500 WBC per microliter, more preferably at least about 2,000 WBC per microliter, even more preferably about 3,000 WBC per microliter, and most preferably about 4,000 WBC per microliter.

[0078] In another embodiment, the vaccine composition of the present invention can be used therapeutically, especially when the microbial infection is localized and / or not life-threatening. In such cases, the vaccine composition of the present invention is administered to a patient suffering from a microbial infection caused by such a microorganism. The vaccine is usually administered intramuscularly to an immunocompetent patient together with a suitable adjuvant to enhance the immune response. Upon administration, effective immunity is generated within about four weeks. If the patient is still infected, the natural immunity generated by the vaccine promotes recovery.

[0079] When so used, the vaccine compositions of the invention are administered in therapeutically effective amounts by any of the accepted modes of administration for agents that serve similar utilities. The actual amount of the vaccine compound, i.e., active ingredient, of the invention will depend on many factors, including the severity of the disease being treated, the age and relative health of the subject, the potency of the vaccine compound used, the route and form of administration, and other factors well known to those skilled in the art.

[0080] An effective or therapeutically effective amount of a vaccine compound of the present invention refers to that amount of vaccine compound that produces antibodies of sufficient titer to ameliorate symptoms or prolong survival in a subject. The toxicity and therapeutic efficacy of such vaccine compounds and vaccine compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.

[0081] The vaccine compositions described herein are typically administered as injectable sterile aqueous compositions containing one or more conventional ingredients well known in the art, including, by way of example only, adjuvants, stabilizers, preservatives, etc.

[0082] combination The vaccine compounds and compositions of the present invention can be used in combination with other therapeutic compounds or other appropriate agents as deemed appropriate by the attending physician. In selected cases, the vaccine compounds of the present invention can be administered simultaneously with antibiotics to treat bacterial infections and agents that enhance the immune response elicited by the vaccine compounds and / or compositions. In the case of antibiotics, the selection of the appropriate antibiotic or antibiotic cocktail and the amount to administer to the patient are well within the skill of the attending physician, based on the specifics of the bacterium in question, the severity of the bacterial infection, and the age, weight, and other relative health conditions of the patient. If necessary, the attending physician can co-administer immune-enhancing drugs or adjuvants in combination with the vaccines described herein.

[0083] The vaccine compositions of the present invention can be administered with an adjuvant to enhance the patient's immune response to the antigen. Adjuvants include, but are not limited to, aluminum compounds such as aluminum hydroxide and aluminum phosphate, and Freund's complete or incomplete adjuvant (e.g., an antigen incorporated into the stabilized water phase of a paraffin oil emulsion). Obviously, paraffin oil can be replaced with other types of oil, such as squalene or peanut oil. Other materials with adjuvant properties include BCG (attenuated Mycobacterium tuberculosis), calcium phosphate, levamisole, isoprinosine, polyanions (e.g., polyA:U), lentinan, pertussis toxin, lipid A, saponin, QS-21, and peptides, such as muramyl dipeptide, and immunostimulatory oligonucleotides, such as CpG oligonucleotides. Rare earth salts, such as lanthanum and cerium, can also be used as adjuvants. The amount of adjuvant used will depend on the subject being treated and the particular antigen being used, and can be readily determined by one of ordinary skill in the art. [Example]

[0084] The present invention will be further understood by reference to the following examples of the invention, which are intended to be purely illustrative. The present invention is not limited in scope by the exemplified embodiments, which are intended only as illustrations of single aspects of the invention. Any methods that are functionally equivalent are within the scope of the present invention. Various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications fall within the scope of the appended claims.

[0085] The following terms are used herein and have the following meanings: If not defined, abbreviations have their conventionally recognized definitions. Å = angstrom aq.=aqueous solution Biotage=Biotage, Div. Dyax Corp., Charlottesville, Virginia, USA bp=boiling point CAD = Charged Aerosol Detector DCM = dichloromethane deg = degrees DMSO = dimethyl sulfoxide eq.=equivalent EtOAc = ethyl acetate FEP = Fluorinated Ethylene Propylene g = grams H 1 -NMR = proton nuclear magnetic resonance h=time HDPE = High Density Polyethylene HPLC = High-Performance Liquid Chromatography MeCN = acetonitrile kg = kilograms mbar = millibar MeOH = methanol mg = milligram mL = milliliters mM = millimolar concentration mmol = millimolar N=normality NBS = N-bromosuccinimide NIS = N-iodosuccinimide NMT = N-methyltryptamine PP = Polypropylene qHNMR = quantitative proton nuclear magnetic resonance RBF = round bottom flask RO = Reverse Osmosis SEC HPLC = Size Exclusion Chromatography HPLC SIM = Secondary Ion Mass TCEP = Tris(2-carboxyethyl)phosphine TLC = thin layer chromatography TMSOTf = methanesulfonic acid, 1,1,1-trifluoro-, trimethylsilyl ester TT = tetanus toxoid μL = microliter μm = microns w / w = weight to weight w / v = weight to volume [Example 1]

[0086] Tetanus Toxoid Stepwise Filtration A sample of crude tetanus toxoid preparation containing monomeric and dimeric toxoids is first passed through a 3- to 5-micron filter to remove higher-order oligomers. This can be done by gradually decreasing the filter pore size. Thus, the toxoid preparation can be passed through a 5-micron filter, followed by a 3-micron filter. Alternatively, the toxoid preparation can be passed through a 5-micron filter, followed by a 4-micron filter, followed by a 3-micron filter. The effectiveness of the 5-micron filtration is assessed by light scattering techniques, which can be used to detect the presence of higher-order oligomers. If necessary, additional stepwise filtrations can be performed to further remove higher-order oligomers. The resulting filtrate contains monomeric and dimeric toxoids. After the oligosaccharide attachment chemistry is fully purified, the filtrate is passed through a 2.5-micron filter, separating the monomeric and dimeric toxoids as a filter cake, while low-molecular-weight impurities pass through with the filtrate. After each filtration step (high and low molecular weight), a filter cake rinse can be performed. [Example 2]

[0087] Attachment of SBAP to TT monomers Step 1: Preparation of N-BABA:

[0088] [ka]

[0089] Commercially available beta-alanine, compound 1, is converted to N-BABA (bromoacetyl-β-alanine), compound 2, by reacting it with at least a stoichiometric amount of commercially available bromoacetyl bromide. In a first vessel, β-alanine is combined with water along with sodium bicarbonate or other suitable base to remove the acid generated during the reaction. The aqueous solution is mixed at approximately 20±5°C until a solution is obtained. The solution is then maintained at approximately 5±5°C. In a separate vessel, the required amount of bromoacetyl bromide is added, followed by dichloromethane. The contents of both vessels are combined. After the reaction is complete, 6N HCl is added and mixed until the pH is approximately 2. The resulting N-BABA is extracted from the solution with a suitable solvent, such as ethyl acetate. The organic layer is concentrated under conventional conditions, such as under reduced pressure at an elevated temperature, such as 60°C. Heptane is then added to precipitate the N-BABA, which is collected on a filter and dried in a vacuum oven at 40°C. This product is used directly in the next step.

[0090] Step 2: Preparation of SBAP:

[0091] [ka] N-BABA, i.e., compound 2, is reacted with N-hydroxysuccinimide (NHS) under conventional conditions known in the art to produce SBAP, i.e., compound 3. Specifically, N-BABA is combined with at least a stoichiometric amount of NHS in a suitable inert solvent, such as methanol, ethanol, or isopropanol. The resulting solution is stirred at approximately 20±5°C until a clear solution is obtained. N-diisopropylcarbodiimide is then added to the reaction mixture and mixed to form a solid. The system is then cooled to 0±5°C, and the resulting SBAP is obtained by filtration. Further purification requires pre-cooling a mixture of isopropanol and heptane, washing the filter cake, and then drying the wet cake in a vacuum oven at approximately 30°C. The resulting SBAP is then used directly in the coupling reaction with TT monomer.

[0092] Alternatively, SBAP can be prepared in the manner described in U.S. Patent No. 5,286,846, which is incorporated herein by reference in its entirety. Specifically, the method described therein is provided by the following synthetic scheme:

[0093] [ka]

[0094] Step 3 - Conjugation As described above, purified TT monomer contains 43 lysine residues / mol, as quantified by free amine assay. Reaction of TT monomer with increasing concentrations of SBAP, from 0 to 170 molar equivalents, resulted in a corresponding decrease in free amine content over the range of 15–110 molar equivalents of SBAP. Steady-state conversion was achieved by loading >110 equivalents of SBAP. Assuming that the loss of free amines is directly proportional to the loading of the SBAP linker, the linker density at saturation was estimated to be 43 moles of SBAP / TT monomer. The monomer / aggregate content of the linker-TT / monomer intermediate and the protein concentration at each titration point were also assessed. The monomer content before linker addition was 99.7%, and increasing the amount of SBAP linker did not significantly change the monomer level (no aggregates were detected). Protein recovery throughout the titration process was also similar. Based on this collective data, a value of 110 molar equivalents of SBAP for 1 h at ambient temperature was selected as the appropriate reaction condition for all subsequent syntheses. [Example 3] Oligosaccharide synthesis

[0095] Building Block Synthesis The following reaction schemes illustrate the synthetic steps used to prepare compounds 3, 5 and 8, which are detailed below.

[0096] [ka]

[0097] Synthesis of Compound D: Commercially available 1,3,4,6-tetra-O-acetyl-2-deoxy-2-N-phthalimido-β-D-glucopyranoside, Compound C (120.6 g, 252.6 mmol), and toluene (200 mL) were placed in a 1 L Buchi flask and rotated at 40 °C until dissolved (<5 min). The solvent was evaporated to give a foam. Toluene (200 mL) was charged to the flask and rotated at 40 °C until dissolved (<5 min). The solvent was evaporated again to dryness. A crystalline solid formed and adhered to the walls. Dichloromethane (800 mL) was added to the flask and rotated around until dissolved; the resulting dark brown solution was charged to a 5 L jacketed reactor, and the flask was rinsed with additional dichloromethane (200 mL) to initiate the reaction. The heating / cooling jacket was set to 20 °C, and the contents of the reactor were mechanically stirred. Ethanethiol (40 mL, 540 mmol) was dissolved in 50 mL of dichloromethane and added to the vessel, and the flask was rinsed with 50 mL of dichloromethane. Boron trifluoride diethyl diethyl etherate (50 mL, 390.1 mmol) was dissolved in dichloromethane (50 mL) and added to the reactor, rinsed with dichloromethane (50 mL) and added to the vessel. The mixture was stirred at 20 °C for 2 hours. The reaction was checked for residual C by TLC. The mobile phase was toluene:ethyl acetate (3:1, v / v), product Rf ≈0.45, C Rf ≈0.3, UV visualization. If a significant amount of C was present, the reaction time needed to be extended.

[0098] Stirring was set to high speed and 4 M aqueous sodium acetate (1.25 L, 5100 mmol) was added. The phases were mixed well for 30 minutes. The pH of the aqueous layer was checked with a dipstick and found to be approximately pH=7. Stirring was stopped and the reaction mixture was allowed to stand for 70 minutes.

[0099] The layers were separated and collected. The organic layer (bottom, 1.2 L) and ethanol (840 mL, 14,400 mmol) were charged to the reactor. The jacket was set to 60°C and the solvent was distilled at atmospheric pressure (dichloromethane bp 40°C and ethanethiol bp 35°C, receiving flask in ice bath). When the distillation slowed, the jacket temperature was increased to 70°C. After 1,300 mL of distillate had been collected, a sample of the vessel contents was taken to determine the ratio of dichloromethane to ethanol. 1 H-NMR confirmed less than 10 mol% dichloromethane. If more dichloromethane was present, further distillation was necessary. Additional ethanol (400 mL) was added, followed by seed crystals of D. The jacket was cooled to 5°C over 30 minutes. The crystal slurry was stirred at 5°C for 3 days. The solid was collected in a sinter funnel and washed with petroleum ether (60-80°C): 1 x 500 mL slurry, 1 x 300 mL plug. The solid was transferred to a 500 mL RBF and dried to constant weight (approximately 4+ hours) on a rotary evaporator (bath temperature 45°C) to give an off-white solid. Expected yield: approximately 86 g (71% from C).

[0100] Synthesis of Compound 1 Anhydrous methanol (33 mL) was charged to a 50 mL round-bottom flask. Sodium methoxide in methanol (30% solution, 25 μL, 0.135 mmol) was added, and the resulting solution was stirred at ambient temperature for 5 minutes. Ethyl 3,4,6-tetra-O-acetyl-2-deoxy-2-N-phthalimido-β-thio-D-glucopyranoside (Compound D) (3.09 g, 6.44 mmol) was added in portions over 10 minutes (approximately 200 mg) at a rate sufficient to dissolve the solid during the addition. The reaction was stirred at ambient temperature for 2.5 hours. TLC (EtOAc) indicated complete consumption of Compound D (Rf = 0.9) and the formation of one more polar spot: Rf = 0.5. A sample was taken and submitted for HPLC completion IPC (2.5 μL of the reaction mixture in 0.8 mL of acetonitrile and 0.2 mL of water), and the pass condition was NMT 1.00 area % Compound D. Acetic acid was added (8 μL, 0.1397 mmol). The pH was checked with a dipstick and confirmed to be approximately pH = 5-6. The mixture was concentrated to near dryness on a rotary evaporator (50 °C). EtOAc (15 mL) was added and the majority of the residue evaporated. The residue was dissolved / slurried in 15 mL of EtOAc and removed from the rotary evaporator. 2 mL of petroleum ether was added and the mixture was stirred at ambient temperature. The crystal slurry was stirred overnight. The solid was collected on a sinter funnel, washed with petrol (2 × 10 mL), and dried to constant weight on a rotary evaporator (bath temperature 45 °C). Expected yield: 1.94 g (85% from compound D).

[0101] Synthesis of compound 2 Compound 1 (2.040 g) was dissolved in pyridine (28 mL), and the solution was concentrated to approximately half its volume (approximately 14 mL) on a rotary evaporator at a bath temperature of 40 °C, yielding a yellow solution. Additional pyridine (14 mL) was added, and the solution was concentrated to approximately 14 mL in the same manner. The solution was placed under argon, and trityl chloride (2.299 g, 1.36 equiv.) was added. After this, an air-cooled condenser was attached, and the solution was heated to 50 °C with stirring. After 4 h, an IPC was performed (HPLC; 5 μL in 800 μL MeCN, residual compound 1 NMT 3.00 area %). Once the IPC was full, the reaction was cooled to 10–15 °C. Benzoyl chloride (1.60 mL, 2.34 equiv.) was added dropwise over 20 min, maintaining the reaction temperature below 20 °C. After the addition was complete, the reaction was allowed to warm to ambient temperature and stirred for at least 3 h. At this point, an IPC was performed (HPLC; 5 μL in 1500 μL MeCN, total NMT 3.00% by area of ​​residual mono-Bz derivative of compound 1). Once the IPC was full, the reaction was cooled to 0° C. and quenched by the slow addition of methanol (0.8 mL), ensuring the reaction temperature remained below 20° C. The quenched reaction was then warmed to ambient temperature.

[0102] The product mixture was diluted with toluene (20 mL) and stirred at ambient temperature for 1 hour before filtering through a sintered funnel to remove the precipitate. The toluene solution was then washed with citric acid (20% w / w, 4 x 20 mL) followed by saturated NaHCO3 (9% w / v, 20 mL), which reacted only slightly with any residual citric acid present. The toluene (top) layer was then washed with brine (20 mL) before being evaporated on a rotary evaporator at a bath temperature of 40 °C to give a yellow / orange syrup (6.833 g). The syrup was subjected to IPC (H 1 NMR, acceptable condition NMT 30 wt% residual toluene). Expected yield: about 6.833 g (147%).

[0103] Synthesis of compound 3 Glacial acetic acid (648 mL) and ultrapure water (72 mL) were mixed together to obtain a 90% acetic acid solution. A portion of the acetic acid solution (710 mL) was added to crude compound 2 (111 g) along with a stir bar. An air-cooled condenser was attached to the flask, and the mixture was then heated to 70 °C. Because 2 was viscous, the mixture only fully dissolved after 1 hour and 20 minutes, at which point stirring was initiated. After 2 hours, an IPC was performed (HPLC; 5 μL in 800 μL MeCN, residual compound 2 NMT 3.00 area %). Once the IPC met specifications, the reaction was cooled to ambient temperature. The mixture was transferred to a sinter funnel, and the precipitated trityl alcohol (31.09 g) was filtered off using house vacuum. The flask was rinsed with additional 90% acetic acid (40 mL), and the entire wash was transferred to a mixing vessel. Toluene (700 mL) and water (700 mL) were added and mixed thoroughly. The aqueous (bottom) layer was a cloudy white solution and was tested for pH (expected to be <2). Two more water washes were repeated (2 × 700 mL; pH ≈2.4 and ≈3, respectively, clear, colorless solutions). Saturated NaHCO3 (9% w / v, 700 mL) was added to the mixing vessel, resulting in a slight reaction (gas evolution). The toluene (top) layer was then washed with brine (700 mL) and then evaporated on a rotary evaporator at a bath temperature of 40 °C to give a yellow / orange solid-liquid mixture (86 g). This mixture was dissolved in 400 mL of toluene (300 mL + 100 mL of wash) and loaded onto a silica column (450 g silica) equilibrated with 3 column volumes (CV) of petroleum ether:toluene (1:1, v:v). The column was eluted using a stepwise gradient, collecting 1 CV (790 mL) fractions. The gradient used was as follows: 4 vol% ethyl acetate:toluene in petroleum ether (1:1 v:v, 4 CV) 8 vol% ethyl acetate:toluene in petroleum ether (1:1 v:v, 12 CV) 15 vol% ethyl acetate:toluene in petroleum ether (1:1 v:v, 4 CV) 20 vol% ethyl acetate:toluene in petroleum ether (1:1 v:v, (4 CV) 30 vol% ethyl acetate:toluene in petroleum ether (1:1 v:v, 1 CV)

[0104] The product eluted over 14 fractions. TLC was used to identify the location of product-containing fractions. All fractions were subjected to IPC (HPLC, NMT 1.50 area % peak at 10.14 min and NMT 1.50 area % peak at 10.94 min). Fractions not satisfying IPC were saved for processing to compound 4. The combined fractions were evaporated on a rotary evaporator at a bath temperature of 45° C. to give a colorless syrup. Expected yield: approximately 60 g (78%).

[0105] Synthesis of compound 4 Crude compound 3 (39.54 g, containing approximately 21 g of compound 3, approximately 37 mmol, collected immediately prior to chromatography) was dissolved in toluene (7.2 mL) and dry pyridine (14.2 mL, 176 mmol, approximately 4.8 equiv.) was added to obtain a homogeneous solution. 7.2 mL of acetic anhydride (76 mmol, approximately 2.1 equiv.) was added, and the mixture was stirred at 25 °C for 18 h. During precipitation of the reaction solid, some of this precipitate was likely compound 4. The reaction was sampled for IPC, and if the amount of compound 3 detected was >1.00 area %, additional dry pyridine (1.4 mL, 17 equiv.) was added and the reaction was continued until the remaining compound 3 was ≤1.00 area % in the liquid phase.

[0106] The reaction was diluted with dichloromethane (112 mL), followed by the addition of water (2.8 mL) and methanol (2.8 mL). The mixture was stirred at 25°C for 3 hours. This stirring period proved sufficient to quench excess acetic anhydride. The mixture was washed with citric acid monohydrate / water 20 / 80 w / w (112 mL). The aqueous phase was back-extracted with dichloromethane (50 mL). The dichloromethane used for back-extraction was set aside and used to back-extract the aqueous phase from the remaining citric acid wash. The main dichloromethane extract was returned to the vessel, and the citric acid wash process was repeated until the pH of the aqueous phase was ≤2 (typically two additional washes). The combined citric acid wash was back-extracted. The back-extract and main dichloromethane extract were then combined. The resulting dichloromethane solution was washed with 5% w / v NaHCO3 (100 mL), and the dichloromethane phase was collected and washed with water (100 mL). The dichloromethane phase was transferred to an evaporation vessel, ethyl acetate (50 mL) was added and the solution was concentrated to a syrup.

[0107] Ethyl acetate (150 mL) was added and heated to 55°C with stirring to dissolve the product. Petroleum ether 60-80 (200 mL) was added and the solution was reheated to 55°C and held for 5 minutes. The solution was cooled to 45°C, seed crystals (30 mg) were added, and then cooled to 18°C ​​with stirring over 3 hours and held at 18°C ​​for at least 1 hour. The crystals were collected by filtration and washed with ethyl acetate / petroleum ether (1 / 2 v / v, 60 mL). Drying under vacuum gave compound 4 (16.04 g, 77% from 2). Expected yield: 16.0 g (77% from compound 2).

[0108] Synthesis of compound 3.1 3-Aminopropan-1-ol (7.01 g, 93 mmol) was dissolved in DCM (70 mL) and cooled to 0 °C. Benzyl chloroformate (5.40 mL, 32 mmol) was dissolved in DCM (20 mL) and added dropwise, maintaining the internal reaction temperature below 10 °C. Upon completion, the flask was stirred at room temperature for 2 h. A sample removed for NMR analysis (IPC: 20 μL + 0.6 mL d6-DMSO) indicated that the benzyl chloroformate reagent had been consumed. The product mixture was then washed with citric acid (10% w / w, 2 × 90 mL), water (90 mL), and brine (90 mL). The DCM (bottom) layer was then rotary evaporated at a bath temperature of 40 °C to yield a slightly cloudy oil / liquid (6.455 g). This oil was dissolved in ethyl acetate (7 mL), warmed to 40 °C as needed to dissolve precipitated solids, and then cooled to room temperature. Petroleum ether (4 mL) was slowly added to the stirred solution along with a seed crystal, at which point the product slowly began to crystallize. Once most of the product had precipitated, the final portion of petroleum ether (17 mL) was slowly added (total solvent added: ethyl acetate:petroleum ether 1:3, 21 mL). The product was then filtered under reduced pressure and washed with petroleum ether (5 mL) to give the product as a fine white powder (4.72 g). Expected yield: approximately 4.7 g (61%).

[0109] Synthesis of compound 5 Compound 4 (1.05 g, 1.73 mmol) was dissolved in dry acetone (12 mL, 0.06% w / w water) and water (39 μL, 2.15 mmol, 1.3 equiv.) at ambient temperature. The solution was then cooled to −10°C. NBS (0.639 g, 3.59 mmol, 2.08 equiv.) was added in one portion. An exotherm of approximately +7°C was expected, and the solution was then immediately recooled to −10°C. 15 min after the NBS addition, the reaction mixture was subjected to IPC (HPLC, pass criteria: less than 2.00 area % of compound 4 remaining). If the reaction was not complete, 1.00 equiv. of NBS (0.307 g, 1.73 mmol, 1.00 equiv.) was added in one portion, and the reaction was held at −10°C for an additional 15 min, followed by another IPC run. The reaction was quenched by the addition of aqueous NaHCO3 (5% w / v, 5 mL). Cooling was discontinued, and the mixture was allowed to warm to 10–20 °C during the following addition. After stirring for 3–5 min, additional aqueous NaHCO3 (5% w / v, 5 mL) was added, and stirring was continued for 5 min. A final aliquot of aqueous NaHCO3 (5% w / v, 10 mL) was added with stirring, followed by sodium thiosulfate (20% w / v, 5 mL). The mixture was stirred for 20 min at 10–20 °C, and then the solid was collected by filtration. The vessel was rinsed on the filter pad with NaHCO3 (5% w / v, 25 mL), and this rinse was filtered off. The filter cake was then rinsed successively with NaHCO3 (5% w / v, 25 mL), then water (25 mL). The (still wet) filter cake was dissolved in DCM (20 mL) and washed with two portions of NaHCO (5% w / v, 20 mL), then once with water (20 mL). The dichloromethane layer was dried by rotary evaporation and then dissolved in ethyl acetate (36 mL) at 65 °C. Petroleum ether 60-80 (10 mL) was then added slowly with stirring, and the mixture was cooled to 45 °C and stirred at 45 °C for 30 min. Additional petroleum ether 60-80 (22 mL) was added with stirring, and the stirred mixture was cooled to 15 °C over 2 h. The product was collected by filtration, washed with petroleum ether / ethyl acetate 2 / 1 v / v (20 mL), and then dried under reduced pressure to give compound 5 (0.805 g, 83% yield; combined purity of α and β anomers by HPLC was 98%).

[0110] Synthesis of compound 7: Compound 4 (500 mg) and Intermediate 3.1 (211 mg, 1.2 equiv.) were weighed into a dry flask, toluene (5 mL) was added, and the solution was concentrated on a rotary evaporator (bath temperature 45 °C). This was repeated once more, after which the starting material was concentrated from anhydrous DCM (5 mL). Once all solvent was removed, the remaining solid was dried under reduced pressure for 10 min. After drying, the starting material was placed under argon, dissolved in anhydrous DCM (5.0 mL), and activated 4 Å molecular sieves (450 mg, pellets) were added. At this point, the NIS reagent was placed under high vacuum to dry. After 10 min, dry NIS (400 mg, 2.0 equiv.) was added, and the solution was stirred at room temperature for 30 min. TMSOTf (8 μL, 5 mol%) was then added quickly, causing the solution to change color from red / orange to dark red / brown. The reaction temperature also increased from 22 °C to 27 °C. Immediately after the addition of TMSOTf, an IPC was performed for reference (HPLC; 10 μL in 1 mL MeCN-HO (8:2)). The reaction was then quenched by the addition of pyridine (20 μL, 0.245 mmol) and stirred at ambient temperature for 5 min. The DCM solution was filtered to remove the molecular sieves, washed with 10% NaSO (3 × 5 mL), brine (5 mL), and concentrated on a rotary evaporator (40 °C bath temperature) to give crude compound 7 as a foamy yellow oil (616 mg). Expected yield: approximately 616 mg (99%).

[0111] Synthesis of compound 8: Crude compound 7 (16.6 g) was dried by evaporation from toluene (2 × 30 mL) and then anhydrous DCM (30 mL) to produce a yellow foam / oil. The flask was then placed under an argon atmosphere, and anhydrous DCM (100 mL) and dry MeOH (260 mL) were added, and the mixture was stirred. The flask was then cooled to 0 °C. Acetyl chloride (3.30 mL, 2.0 equiv.) was added dropwise, maintaining the internal temperature below 10 °C. Upon completion of the addition, the mixture was stirred at ambient temperature for 16 h. At this point, an IPC was performed (HPLC; 20 μL in 1 mL MeCN, residual compound 7 was less than 3 area %). The flask was then cooled to 0 °C, and the pH of the product solution was adjusted to pH 6.5-7.5 by adding N-methylmorpholine (a total of 7.0 mL required). The product mixture was diluted with DCM (50 mL) and washed with HO (2 × 200 mL). The second HO wash was cloudy and contained the target material by TLC, so it was back-extracted with DCM (50 mL). The combined DCM layers were then washed with brine (8 mL) and then rotary evaporated at a bath temperature of 40 °C to give an off-white foam / oil (approximately 16.8 g). This mixture was dissolved in 140 mL of toluene (100 mL + 40 mL wash) and loaded onto a silica column (85 g silica) equilibrated with 3 column volumes (CV) of 30 vol% ethyl acetate in petroleum ether. The column was eluted using a stepwise gradient, collecting 1 CV (140 mL) fractions. The gradient used was as follows: 30 vol% ethyl acetate in petroleum ether (3 CV) 35 vol% ethyl acetate in petroleum ether (4 CV) 40 vol% ethyl acetate in petroleum ether (9 CV) 50 vol% ethyl acetate in petroleum ether (4 CV) 60 vol% ethyl acetate in petroleum ether (3 CV) The product eluted over 12 fractions. All fractions were subjected to IPC (HPLC, impurity peak with NMT 1.50 area % at 230 nm). The combined fractions were evaporated on a rotary evaporator at a bath temperature of 40° C. to give an off-white foam, which solidified to give 8 as a crisp solid (10.45 g). Expected yield: 10.45 g (66%). [Example 4]

[0112] Synthesis of disulfide (compound 17)

[0113] [ka]

[0114] compound 17 The overall synthetic procedure for synthesizing compound 17 is described in the synthetic scheme below.

[0115] [ka]

[0116] Synthesis of compound 9: Compound 5 (1620 g, 1.18 equiv.) and toluene (18 kg) were charged in this order to a 50 L buchi bowl. The bowl was warmed in a water bath set at 50 ± 10 °C for 30 minutes. Evaporation was carried out under reduced pressure using a water bath temperature of 50 ± 10 °C until no more solvent was distilled. The water bath was cooled to 20 ± 10 °C. Trichloroacetonitrile (7.1 kg, 21 equiv.) and dry DCM (6.5 kg) were charged to the bowl under a nitrogen atmosphere. A suspension of sodium hydride (5.6 g, 0.060 equiv.) in dry DCM (250 g) was charged to the bowl under a nitrogen atmosphere. The contents of the bowl were mixed by rotation at a water bath temperature of 20 ± 10 °C for 1 to 2 hours. Compound 5 dissolved during the reaction. The contents of the bowl were sampled and the reaction completion IPC (H 1NMR was performed, integrating the triplet peak at 6.42 ppm (product) compared to 6.35 ppm (starting material); acceptance criteria: ≤5% residual starting material. Compound 3 (1360 g, 2.35 mol), dry DCM (12.3 kg), and powdered 4Å molecular sieves (136 g) were charged, in that order, to a 50 L reactor. The reactor contents were mixed for 24 h. The reactor contents were sampled with a syringe filter and analyzed by Karl Fischer (AM-GEN-011, acceptance criteria: ≤0.03% w / w). After the moisture threshold was reached (approximately 24 h), the reactor contents were adjusted to 0 ± 5 °C. The contents of the Buchi bowl were transferred to the reactor header as volume permitted. A solution of trimethylsilyl trifluoromethanesulfonate (100 g, 0.18 equiv.) in dry DCM (1250 g) was charged to the reactor under a nitrogen atmosphere. The contents of the header were discharged into the reactor, and the reactor contents were maintained at 0±10°C during the addition. The addition took 15-20 minutes. Dry DCM (1250 g) was charged to the buchi bowl and transferred to the reactor header. The contents of the header were discharged into the reactor, and the reactor contents were maintained at 0±10°C during the addition. The reactor contents were stirred at 0±5°C for 60 minutes. The reactor contents were sampled for reaction completion using IPC (HPLC, pass criterion ≤5% starting material). The reaction was quenched by charging N-methylmorpholine (85 g, 0.36 equiv.) to the reactor. The reactor contents were sampled for quench completion using IPC (wet pH paper, pass criterion ≥ pH 7). Silica gel (4.9 kg) was charged to the buchi bowl. The reactor contents were transferred to the buchi bowl. Evaporation was carried out under reduced pressure using a water bath temperature of 40±10°C until no more solvent was distilled. Silica gel (1.4 kg) was loaded into the Buchi bowl, followed by rinsing the reactor with dichloromethane (7.0 kg). The contents of the bowl were swirled to prevent solids from adhering to the surface of the bowl. Evaporation was carried out under reduced pressure using a water bath temperature of 40±10°C until no more solvent was distilled. The contents of the bowl were divided into three portions for silica gel chromatography. A 150LKP-SIL cartridge was installed in the Biotage system.Ethyl acetate (7.8 kg) and petroleum ether (22 kg) were charged to a 50 L reactor along with one-third of the silica gel-adsorbed reaction mixture, mixed thoroughly, and then transferred to the Biotage solvent reservoir. The contents of the solvent reservoir were eluted through the column to condition it. The eluate was collected in a 20 L jerry can and discarded. The column was run in three batches, each eluted with ethyl acetate / petroleum ether as described below.

[0117] Ethyl acetate (1.6 kg) and petroleum ether (4.4 kg) were charged to a Biotage solvent reservoir and mixed thoroughly before eluting from the column. The column effluent was collected in a 20 L jerry can.

[0118] Ethyl acetate (25 kg) and petroleum ether (26 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can.

[0119] Ethyl acetate (31 kg) and petroleum ether (22 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 5 L glass lab bottle.

[0120] Ethyl acetate (16 kg) was charged to the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 20 L jerry can.

[0121] The remaining two portions of dry loaded silica were prepared and the columns were repeated as above.

[0122] Column fractions were sampled for product purity (TLC [10% acetone in toluene, Rf 0.5]) to identify fractions containing product. The accepted column fractions were combined and placed in a 100 L buchi bowl. Toluene was used to rinse the crystalline material from the accepted fraction container into the bowl. Evaporation was carried out under reduced pressure using a water bath temperature of 40 ± 10 °C until no more solvent was distilled. Toluene (1.7 kg) was charged to the bowl and the contents were swirled until the solids dissolved. t-Butyl methyl ether (4.4 kg) was charged to the bowl over 20-40 minutes. The contents of the bowl were swirled at a temperature of 20 ± 5 °C for 12-24 hours. The contents of the bowl were evaporated to 6 L. The mixture was transferred to a Nutsche filter and the solvent removed by vacuum filtration. t-Butyl methyl ether (620 g) was charged to a bowl, transferred to the Nutsche filter, and filtered through the filter cake. The filter cake was air-dried in the filter and then transferred to a vacuum oven and dried under reduced pressure at a setting of 30 °C to remove residual solvent. The solid was sampled for analysis and retention. The solid was transferred to a screw-cap Nalgene container and stored at or below -15 °C. Expected yield: 1.68-1.94 kg of compound 9 (65-75%).

[0123] Synthesis of compound 10: The reagents were prepared as follows: N-iodosuccinimide (241 g, 2.20 equiv.) was dried under reduced pressure in a vacuum oven set at 30° C. for 24 hours. A solution of sodium chloride (300 g) in water (3000 g) was prepared in a 5 L lab bottle. A solution of sodium thiosulfate (1100 g) in water (6000 g) was prepared in a 50 L reactor and divided into two portions.

[0124] Compound 8 (355 g, 0.486 mol) and compound 9 (634 g, 1.10 equiv.) were charged to a 20 L buchi bowl, followed by toluene (1500 g) and heated at 40 ± 5°C until dissolved. Evaporation was carried out under reduced pressure using a water bath temperature of 35 ± 10°C until no more solvent was distilled. Toluene (1500 g) was charged to the buchi bowl. Evaporation was carried out under reduced pressure using a water bath temperature of 35 ± 10°C until no more solvent was distilled. Dry dichloromethane (4000 g) was charged to the buchi bowl. The bowl was swirled until the solids dissolved, and the solution was transferred to a 5 L reactor with a jacket temperature of 20 ± 5°C. Dry dichloromethane (710 g) was charged to the buchi bowl. The bowl was swirled to rinse the bowl surface, and the solution was transferred to the 5 L reactor. The contents of the reactor were mixed in a 200 mL flask with a 200 mL ethanol solution at a reagent ratio of IPC (H 1 The contents of the reactor were sampled for reaction completion (HPLC). Dry N-iodosuccinimide was charged to the reactor under a nitrogen atmosphere, and the reactor was stirred for 5-15 minutes. The contents of the reactor were adjusted to 20°C ± 3°C. Trimethylsilyl trifluoromethanesulfonate (5.94 g, 0.055 eq) in dry DCM (60 g) was charged to the reactor over 5-15 minutes, maintaining the temperature of the contents at 20°C ± 3°C. The reaction mixture was stirred at 20°C ± 3°C for 20 ± 3 minutes. The contents of the reactor were sampled for reaction completion (HPLC). N-methylmorpholine (98 g, 2 eq) was charged to the reactor and mixed thoroughly. One portion of the sodium thiosulfate solution prepared above was charged to a 50 L reactor. 5 L of the reactor contents were transferred to the 50 L reactor containing the sodium thiosulfate solution and mixed thoroughly. The bottom layer was drained into an HDPE jerry can.

[0125] DCM (570 g) was charged to a 5 L reactor along with the top layer from the 50 L reactor and mixed thoroughly. The bottom layer was combined with the bottom layer from the previous HDPE jerry can. The top layer was transferred to another HDPE jerry can and held until yield was confirmed. The combined organic phase (bottom layer) was charged to a 50 L reactor, followed by another portion of sodium thiosulfate and mixed thoroughly. The bottom layer was drained into an HDPE jerry can. The top layer was held in the HDPE jerry can until yield was confirmed. The sodium chloride solution was charged to a 50 L reactor along with the organic phase (bottom layer) and mixed thoroughly. Silica gel (1300 g) was charged to a Buchi bowl and attached to a rotary evaporator. The bottom layer of the reactor was charged to the Buchi bowl. The contents of the bowl were swirled to prevent adsorption to the bowl, and evaporation was carried out under reduced pressure using a water bath temperature of 40 ± 5 °C until no solids distilled over. The contents of the bowl were divided into two equal portions. Silica gel (200 g) was charged into a Buchi bowl, followed by dichloromethane (700 g). The contents of the bowl were swirled to prevent solids from adhering to the surface of the bowl. The bowl was evaporated under reduced pressure at a water bath temperature of 40±10°C until no more solvent was distilled. The contents of the bowl were divided into two portions, and one portion was added to each of the previous silica gel samples.

[0126] Each portion was independently purified on silica gel using the following procedure (samples were stored below ≦15°C while awaiting purification): A 150L KP-SIL cartridge was installed in a Biotage system. Ethyl acetate (15.5 kg) and petroleum ether (16.5 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to two Biotage solvent reservoirs. The contents of the solvent reservoirs were eluted through the columns to condition the columns. The eluate was collected in a 20 L jerry can and discarded. A portion of the dry packed silica from above was loaded into a Biotage Sample-Injection Module (SIM) and eluted with ethyl acetate / petroleum ether as follows:

[0127] Ethyl acetate (6.2 kg) and petroleum ether (6.6 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir. The column effluent was collected in a 20 L jerry can.

[0128] Ethyl acetate (19.5 kg) and petroleum ether (19.2 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can.

[0129] Ethyl acetate (13.6 kg) and petroleum ether (12.3 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can.

[0130] Ethyl acetate (14.2 kg) and petroleum ether (11.9 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can.

[0131] Ethyl acetate (29.7 kg) and petroleum ether (22.9 kg) were loaded into the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 20 L jerry can until fraction 11, then in a 5 L HDPE jerry can.

[0132] Ethyl acetate (15.5 kg) and petroleum ether (11.0 kg) were loaded into the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 5 L HDPE jerry can.

[0133] Ethyl acetate (29.7 kg) and petroleum ether (13.2 kg) were loaded into the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 5 L HDPE jerry can.

[0134] Ethyl acetate (15.5 kg) was charged to the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 5 L HDPE jerry can.

[0135] Column fractions were sampled for product purity (TLC to identify fractions containing product). Fractions representing 75-95% area of ​​compound 10 from the first two columns were combined in a Buchi bowl packed with silica gel (400 g) and evaporated under reduced pressure using a water bath temperature of 40 ± 10 °C until no more solvent was distilled. The contents of the bowl were purified as follows: 150L KP-SIL cartridges were installed in a Biotage system. Ethyl acetate (15.5 kg) and petroleum ether (16.5 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to two Biotage solvent reservoirs. The contents of the solvent reservoirs were eluted through the columns to condition them. The eluate was collected in a 20 L jerry can and discarded. The contents of the bowl were loaded into a Biotage Sample-Injection Module (SIM) and eluted with ethyl acetate / petroleum ether as follows:

[0136] Ethyl acetate (6.2 kg) and petroleum ether (6.6 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir. The column effluent was collected in a 20 L jerry can.

[0137] Ethyl acetate (19.5 kg) and petroleum ether (19.2 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can.

[0138] Ethyl acetate (13.6 kg) and petroleum ether (12.3 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can.

[0139] Ethyl acetate (14.2 kg) and petroleum ether (11.9 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can.

[0140] Ethyl acetate (29.7 kg) and petroleum ether (22.9 kg) were loaded into the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 20 L jerry can until fraction 11, then in a 5 L HDPE jerry can.

[0141] Ethyl acetate (15.5 kg) and petroleum ether (11.0 kg) were loaded into the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 5 L HDPE jerry can.

[0142] Ethyl acetate (29.7 kg) and petroleum ether (13.2 kg) were loaded into the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 5 L HDPE jerry can.

[0143] Ethyl acetate (15.5 kg) was charged to the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 5 L HDPE jerry can.

[0144] The accepted column fractions from all three columns were combined in a Buchi bowl and evaporated under reduced pressure using a water bath temperature of 40±10°C until no more solvent distilled. The contents of the bowl were sampled for analysis and retention. The bowl was sealed and transferred to storage at ≤-15°C. Expected yield: 440-540 kg (52-64% yield).

[0145] Synthesis of compound 11 Dichloromethane was charged to a buchi bowl containing compound 10 (635 g, 0.345 mol) (PN0699) and heated at 30 ± 10 °C until dissolved. Methanol (3.2 kg) was charged to the bowl. The contents of the bowl were adjusted to 0 ± 3 °C. Acetyl chloride (54.1 g, 2 equiv.) in dichloromethane (660 g) was charged to the bowl while maintaining the temperature of the contents at 0 ± 10 °C. The contents of the bowl were adjusted to 20 ± 3 °C, and the mixture was stirred for 40-48 h. The contents of the bowl were sampled for reaction completion IPC (HPLC, passed). The contents of the bowl were adjusted to 0 ± 3 °C. N-methylmorpholine (139 g, 4 equiv.) was charged to the bowl and mixed thoroughly. The contents of the bowl were sampled for quench completion IPC (pH paper, passed ≤ pH 7). The contents of the bowl were concentrated under reduced pressure in a 35 ± 10 °C water bath. Ethyl acetate (4.8 kg) and water (5.5 kg) were charged to a buchi bowl and swirled to dissolve the contents of the bowl. The contents of the bowl were transferred to a 50 L reactor and mixed thoroughly. The bottom layer was drained into an HDPE jerry can. The top layer was transferred to a buchi bowl equipped with a rotary evaporator, and the contents were concentrated under reduced pressure in a 35±10°C water bath. The bottom layer of the HDPE jerry can was charged to a 50 L reactor containing ethyl acetate (1.5 kg) and mixed thoroughly. The bottom layer was drained into an HDPE jerry can and held until yield was confirmed. The top layer was transferred to a buchi bowl equipped with a rotary evaporator, and the contents were concentrated under reduced pressure in a 35±10°C water bath. The contents of the bowl were sampled for analysis and retention. The bowl was sealed and transferred to storage at ≤-15°C. Expected yield: 518-633 kg (90-110% yield).

[0146] Synthesis of compound 12: The reagents were prepared as follows: Two portions of N-iodosuccinimide (143 g, 3.90 equivalents) were dried under reduced pressure in a vacuum oven set at 30° C. for 24 hours. A solution of sodium chloride (450 g) in water (1850 g) was prepared in a 5 L lab bottle and divided into two approximately equal portions. A solution of sodium thiosulfate (230 g) in water (2080 g) was prepared in a 5 L lab bottle and divided into four approximately equal portions.

[0147] Compound 9 (504 g, 1.30 equiv.) was charged to a 50 L buchi bowl containing compound 11 (607 g, 0.327 mol), followed by toluene (1500 g) and heated at 40 ± 5 °C until dissolved. Evaporation was carried out under reduced pressure using a water bath temperature of 35 ± 10 °C until no more solvent was distilled. Toluene (1500 g) was charged to the buchi bowl. Evaporation was carried out under reduced pressure using a water bath temperature of 35 ± 10 °C until no more solvent was distilled. Dry DCM (2400 g) was charged to the buchi bowl. The bowl was swirled until the solids dissolved, and half of the solution was transferred to a 5 L reactor with a jacket temperature of 20 ± 5 °C. The remaining half of the solution was transferred to a 5 L lab bottle. Dry DCM (710 g) was charged to the buchi bowl. The bowl was swirled to rinse the bowl surface, and half of the solution was transferred to the 5 L reactor. The remaining half was charged to the 5 L lab bottle described above and stored under nitrogen for use in the second batch. A portion of the dried N-iodosuccinimide was charged to a reactor under a nitrogen atmosphere. The reactor contents were adjusted to -40°C ± 3°C. Trimethylsilyl trifluoromethanesulfonate (9.09 g, 0.25 effective equivalents) in dry dichloromethane (90 g) was charged to the reactor over 15 minutes while maintaining the temperature of the contents at -40°C ± 5°C. The reaction mixture was stirred at -40°C ± 3°C for 30 ± 5 minutes, then adjusted to -30°C ± 3°C and stirred for 150 minutes. The reactor contents were sampled for reaction completion. N-methylmorpholine (33.1 g, 2 effective equivalents) was charged to the reactor and mixed thoroughly. One portion of the sodium thiosulfate solution prepared above was charged to the 5 L reactor and mixed thoroughly. The bottom layer was drained into a 5 L lab bottle. DCM (400 g) was charged to a 5 L reactor and mixed thoroughly. The bottom layer was combined with the previous bottom layer from the 5 L lab bottle. The combined organic phase was charged to a 5 L reactor, followed by another portion of sodium thiosulfate and mixed thoroughly. The bottom layer was drained into a 5 L lab bottle. The upper portion of the sodium chloride solution was charged to the reactor, followed by the contents of the previous lab bottle. The bottom layer from the reactor was charged to a buchi bowl and evaporated under reduced pressure using a water bath temperature of 40 ± 10 °C until no more solvent distilled. The reactor was washed and dried.

[0148] The second portions of compounds 9 and 11 were charged to the reactor and treated identically to the first batch. Following the organic extraction of the second batch, the reaction mixtures were combined in the reactor. The sodium chloride solution portion was charged to the reactor and thoroughly mixed. Silica gel (1700 g) was charged to a buchi bowl and a rotavapor was attached. The bottom layer of the reactor was charged to the buchi bowl and evaporated under reduced pressure using a water bath temperature of 40 ± 10 °C until no more solvent was distilled. The contents of the bowl were divided into two equal portions and purified independently with silica gel. A 150L KP-SIL cartridge was installed in a Biotage system (commercially available from Biotage, a division of Dyax Corporation, Charlottesville, Virginia, USA). Ethyl acetate (7.7 kg) and petroleum ether (22.0 kg) were charged to a 50 L reactor, thoroughly mixed, and then transferred to two Biotage solvent reservoirs. The contents of the solvent reservoir were eluted through the column to condition it. The eluate was collected in a 20 L jerry can and discarded. A portion of the dry packed silica from above was packed into a Biotage Sample-Injection Module (SIM) and eluted with ethyl acetate / petroleum ether as follows:

[0149] Ethyl acetate (1.5 kg) and petroleum ether (4.4 kg) were charged into an HDPE jerry can, mixed thoroughly, and then transferred to the Biotage solvent reservoir. The column effluent was collected in a 20 L jerry can.

[0150] Ethyl acetate (18.6 kg) and petroleum ether (8.8 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can.

[0151] Ethyl acetate (19.2 kg) and petroleum ether (8.4 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can.

[0152] Ethyl acetate (29.7 kg) and petroleum ether (11.9 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can.

[0153] Ethyl acetate (15.5 kg) was loaded into the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 5 L glass lab bottle.

[0154] Column fractions were sampled for product purity (TLC to identify fractions containing product). Fractions representing 75-95% area of ​​compound 12 from the first two columns were combined in a buchi bowl packed with silica gel (400 g) and evaporated under reduced pressure using a water bath temperature of 40 ± 10 °C until no more solvent was distilled. Ethyl acetate (7.7 kg) and petroleum ether (22.0 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to two Biotage solvent reservoirs. The contents of the solvent reservoirs were eluted through the columns to condition them. The eluate was collected in a 20 L jerry can and discarded. The dry packed silica containing the impure product was loaded into a Biotage Sample-Injection Module (SIM) and eluted as detailed below:

[0155] Ethyl acetate (1.5 kg) and petroleum ether (4.4 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir. The column effluent was collected in a 20 L jerry can.

[0156] Ethyl acetate (19.2 kg) and petroleum ether (8.4 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can.

[0157] Ethyl acetate (18.6 kg) and petroleum ether (8.8 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can.

[0158] Ethyl acetate (29.7 kg) and petroleum ether (11.9 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can.

[0159] Ethyl acetate (15.5 kg) was loaded into the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 5 L glass lab bottle.

[0160] Column fractions were sampled for product purity (TLC to identify fractions containing product, HPLC acceptance criteria ≥ 95% compound 12, and > 2.5% no single impurity). Accepted column fractions from all three columns were combined in a Buchi bowl and evaporated under reduced pressure using a water bath temperature of 40 ± 10 °C until no more solvent distilled. The contents of the bowl were sampled for analysis and retention. The bowl was sealed and transferred to storage at ≤ -15 °C. Expected yield: 494-584 kg (52-64% yield).

[0161] Synthesis of compound 13: Glacial acetic acid (7.5 kg) and ethyl acetate (6.5 kg) were combined in a suitable container and labeled "GAA / EA solution." Sodium bicarbonate (0.5 kg) was dissolved in RO water (10 kg) and labeled "5% w / w sodium bicarbonate solution." Palladium on activated carbon (100 g, specifically Johnson Matthey, Aliso Viejo, California, USA, product number A402028-10) and GAA / EA solution (335 g) were charged in that order to a reaction vessel. Compound 12 (270 g) was dissolved in GAA / EA solution (1840 g) and transferred to a 50 L reaction vessel. The solution was purged of oxygen by pressurizing with nitrogen to 10 bar and then released. This was repeated two more times. The contents of the reactor were pressurized under hydrogen to 10 bar and then released. The reaction mixture was hydrogenated at 20 bar H2 for 1.5 days. The pressure was then released and the solution was purged of hydrogen by pressurizing with nitrogen to 10 bar and then released. This was repeated once. The reaction mixture was filtered through a pad of Celite (300 g). The Celite cake was washed with the GAA / EA solution (2 × 5.5 kg). The combined filtrate was evaporated under reduced pressure (bath temperature 40 ± 5 °C). The residue was co-evaporated with ethyl acetate (2.3 kg) in two portions. The expected weight of the crude product was approximately 316 g. A Biotage system was fitted with a 150MKP-SIL cartridge equipped with a 5 L Sample Injection Module (SIM). Ethyl acetate (10.6 kg) and glacial acetic acid (1.4 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir. The contents of the solvent reservoir were eluted through the column to condition it. The eluate was discarded. The crude product was dissolved in ethyl acetate (422 g) and glacial acetic acid (55 g). The resulting solution was loaded onto a SIM and passed through a column. The reaction mixture was chromatographed as follows:

[0162] Ethyl acetate (13.8 kg) and glacial acetic acid (1.8 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir.

[0163] The contents of the solvent reservoir were eluted through the column via the SIM and the eluate was collected in a 20 L jerry can.

[0164] Ethyl acetate (10.3 kg), glacial acetic acid (1.3 kg) and methanol (206 g) were charged to a 50 L reactor, mixed thoroughly and then transferred to the Biotage solvent reservoir.

[0165] The contents of the solvent reservoir were eluted through the column and the eluate was collected in a 5 L jerry can.

[0166] Ethyl acetate (6.6 kg), glacial acetic acid (0.9 kg) and methanol (340 g) were charged to a 50 L reactor, mixed thoroughly and then transferred to the Biotage solvent reservoir.

[0167] The contents of the solvent reservoir were eluted through the column and the eluate was collected in approximately 2.5 L fractions into a 5 L jerry can.

[0168] Ethyl acetate (31.4 kg), glacial acetic acid (4.1 kg) and methanol (3.40 kg) were charged to a 50 L reactor, mixed thoroughly and then transferred to the Biotage solvent reservoir.

[0169] The contents of the solvent reservoir were eluted through the column and the eluate was collected in a 5 L jerry can.

[0170] The fractions containing compound 13 were combined and evaporated under reduced pressure (bath temperature 40±5°C). The residue was dissolved in ethyl acetate (3.1 kg) and washed with 5% w / w sodium bicarbonate solution (9.3 kg) until the pH of the aqueous medium was ≥ 8. The ethyl acetate phase was evaporated under reduced pressure (bath temperature 40±5°C). The contents of the bowl were sampled for analysis and retention. Expected yield: 182-207 g (71-81%).

[0171] Synthesis of compound 16 Dry dichloromethane (2.5 kg) was charged to a buchi bowl containing compound 13 (211 g, 76.5 mmol, 1.00 equiv.) and swirled without heating until dissolved. A solution of (2,5-dioxopyrrolidin-1-yl) 4-acetylsulfanylbutanoate (25.8 g, 99.4 mmol, 1.30 equiv.) in dry dichloromethane (200 g) was added to the buchi bowl. The bowl was swirled at ambient temperature for 1 hour and then concentrated under reduced pressure at a water bath temperature of 40 ± 5 °C. Toluene (0.8 kg) was added to the bowl and removed under reduced pressure twice at a water bath temperature of 40 ± 5 °C. Toluene (0.8 kg) was added to the bowl and dissolved in the residue. Silica gel (557 g) was placed in the reaction vessel, and the solvent was removed under reduced pressure at a water bath temperature of 40 ± 5 °C. A Biotage system was fitted with a 150M KP-SIL cartridge equipped with a 5 L Sample Injection Module (SIM). Toluene (10.1 kg) and acetone (1.0 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir (solvent A). The reaction mixture was purified as follows:

[0172] The column was conditioned by passing solvent A through it and eluting it. The eluate was discarded.

[0173] The dried packed silica gel was transferred to the SIM.

[0174] Toluene (9.6 kg) and acetone (1.5 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir (solvent B).

[0175] Solvent B was eluted through the column and the eluate was collected in a 5 L jerry can.

[0176] Toluene (53.6 kg) and acetone (12.2 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir (solvent C).

[0177] Solvent C is eluted through the column and the eluate is collected in a 5 L jerry can.

[0178] Toluene (8.4 kg) and acetone (2.6 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir (solvent D).

[0179] Solvent D was eluted through the column and the eluate was collected in a 5 L jerry can.

[0180] Toluene (23.4 kg) and acetone (9.2 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir (solvent E).

[0181] Solvent E was eluted through the column and the eluate was collected in a 5 L jerry can.

[0182] Fractions containing compound 16 (acceptance criteria: ≥ 90% compound 16 and > 2.5% absence of a single impurity) were combined and evaporated under reduced pressure (bath temperature 40 ± 5 °C). The residue was dissolved in tetrahydrofuran (4.4 kg) and concentrated under reduced pressure at a water bath temperature of 40 ± 5 °C. The contents of the bowl were sampled for analysis and retention. Expected yield: 169-192 g (76-86%).

[0183] Synthesis of compound 17 Prior to start-up, the reactor was marked at the 2.5 L, 3.5 L, and 3.9 L levels and attached to a vacuum controller. Dichloromethane was charged to a buchi bowl containing 140 g of compound 16 and transferred to the Reactor Ready vessel. The contents of the buchi bowl were transferred to the Reactor Ready vessel using two rinses of DCM (333 g). Ethanol (2.50 kg) was added to the Reactor Ready. The reaction mixture was concentrated to the 2.5 L mark (target vacuum 250 mbar). Ethanol (1.58 kg) was added to the Reactor Ready and concentrated to the 3.5 L mark. The reaction was diluted with ethanol to the 3.9 L mark. The contents of the reactor were placed under inert gas by applying a partial vacuum and venting the nitrogen. A slow flow of nitrogen was maintained throughout the reaction. Hydrazine monohydrate (1.13 kg, 1.11 L) was charged into a 5 L Reactor Ready vessel under a nitrogen atmosphere. The temperature gradient was set as follows: initial temperature 20°C, final temperature 60°C, linear temperature gradient over 50 minutes (0.8 degrees / min), and active control of the reactor contents. The vessel temperature was held at 60°C for 45 minutes. The cooling gradient temperature was set at -2 degrees / min, with a final temperature of 20°C. The contents were drained into suitable HDPE jugs and weighed. Equal amounts were transferred to eight polypropylene centrifuge vessels with FEP encapsulation seals. Each centrifuge vessel was filled with ethanol (750 g) and stirred for 30 minutes at ambient temperature. The vessel was centrifuged (5300 RCF, 15°C, 30 minutes). Residual hydrazine on the outside of the vessel was removed by rinsing the outside of the bottle with acetone and then water before removing from the fume hood. The supernatant from the centrifuge vessel was decanted, and the remaining pellet was dissolved in low-endotoxin water (LE water) (1960 g) and transferred to a 5 L Reactor Ready vessel. The contents were stirred at medium speed for approximately 15-20 minutes every 1.5 hours, while aerating the solution using a sparger. The reaction was then stirred overnight at 20°C in a sealed vessel. The reaction was considered complete when IPC indicated a free pentamer composition of less than 3% (reported total area%). Filtration (using a P3 sintered glass funnel and a 5 L Buchner flask) was required if insoluble material was present in the reaction mixture.The reactor contents were freeze-dried in two Lyoguard trays. The shelf temperature was set at -0.5°C for 16-20 hours, then at 20°C until dry. The freeze-dried product was dissolved in LE water (840 g) and divided equally among six centrifuge bottles. Acetone (630 g) was added to each container after 15 minutes of stirring. Isopropanol (630 g per container) was added to each container and stirring continued for 20 minutes. The contents were centrifuged at 5300 RCF and 15°C for 1 hour. The supernatant was discarded, and LE water (140 g) was added to each container. Each pellet was dissolved in water by agitating the mixture at ambient temperature using an orbital shaker until the pellet was dissolved. Acetone (630 g) was added to each container and stirring continued for 15 minutes. Isopropanol (630 g per container) was added to each container and stirring continued for 20 minutes. The contents were centrifuged at 5300 RCF and 15°C for 1 hour. The supernatant was discarded, and each pellet was dissolved in water by adding LE water (100 g) and stirring at ambient temperature. The solution was transferred to a Lyoguard tray, and the bottles were rinsed with additional LE water (66 g each), with the rinse transferred to the same tray. The product was lyophilized at a shelf temperature of -0.5°C for 16-20 hours, then at 20°C until dry. The lyophilized product was sampled for analysis and storage. The Lyoguard trays were double-wrapped, labeled, and stored in a freezer (≤-15°C). The potency of the lyophilized product was determined using qH NMR. This procedure yielded crude penta dimer 17. Expected yield: 26.1-35.5 g (61-83%).

[0184] The identity of compound 17 was confirmed using a 500 MHz instrument. 1 H and 13 The NMR spectra were determined by C NMR. A reference solution of t-butanol was prepared at 25 mg / mL in DO. Samples were prepared at 13 mg / mL in DO, and the reference solution was added to the sample. The composition of the final test sample was 10 mg / mL pentadimer and 5 mg / mL t-butanol. 1 H and 13 C spectra were acquired and integrated, and the resulting chemical shifts were assigned by comparison with theoretical shifts. 1 H NMR and13 The C NMR spectra are shown in Figures 1 and 2, respectively. [Example 5]

[0185] Conversion of crude pentadimer to the free base form. Amberlite FPA91 (1.46 kg; 40 g / g crude pentadimer—corrected for potency) was loaded into a large column. 8 L of 1.0 M NaOH solution was prepared by adding NaOH (32 g) to LE water (8.00 kg) in a 10 L Schott bottle. This solution was passed through the Amberlite resin over 1 hour. LE water (40.0 kg) was passed through the Amberlite resin. The resin was flushed with additional LE water (approximately 10 kg aliquots) until the flow-through pH was <8.0. Crude pentadimer (49 g, PN0704), stored in a Lyoguard tray, was warmed to ambient temperature. LE water (400 g) was added to the Lyoguard tray containing the crude pentadimer (49 g) and, after complete dissolution, transferred to a 1 L Schott bottle. The tray was rinsed with an additional LE water (200 g), and these washes were added to the contents of the Schott bottle. The crude pentadimer solution was carefully poured onto the top of the resin. A 1 L Schott bottle was rinsed with LE water (200 g) and added to the resin. The Amberlite tap was opened, allowing the crude pentadimer solution to slowly transfer onto the resin over approximately 5 minutes. The tap was stopped, and the material was allowed to sit on the resin for approximately 10 minutes. LE water was poured onto the top of the resin. The tap was opened and elution was performed with LE water, collecting approximately 16 500 mL fractions. Each fraction was analyzed by TLC charring (10% H2SO4 in EtOH). All carbohydrate-containing fractions were combined and filtered through a Millipore filter using a 0.2 μm nylon filter membrane. The solution was divided evenly into 5-6 Lyoguard trays. The filter vessel was rinsed with LE water (100 g) and divided between the trays. The material was lyophilized within the trays. The shelf temperature was set to -10°C for 16-20 hours, then to +10°C until the material was dry. LE water (150g) was filled into all but one of the Lyoguard trays and transferred to the remaining tray containing the dried material. Each empty tray was rinsed with more LE water (100g) and this rinse was added to the final Lyoguard tray. The final Lyoguard tray was freeze-dried. The shelf temperature was set to -10°C for 16-20 hours, then +10°C until the material was dry.The product was sampled for analysis and retention. The dried material was transferred to HDPE or PP containers and stored at ≦−15°C. Expected yield: 31-34 g (86-94%).

[0186] TCEP reduction of the disulfide bond in the dimer is rapid and nearly stoichiometric. Using stoichiometric reduction with TCEP, approximately two equivalents of glucosamine pentasaccharide monomer were obtained. Specifically, the pentasaccharide dimer was dissolved in a reaction buffer (50 mM HEPES buffer, pH 8.0) containing 1 molar equivalent of TCEP. After 1 h at ambient temperature, the reaction was analyzed by HPLC with CAD detection. Under these conditions, the conversion to pentaglucosamine monomer (peak at approximately 10 min) was nearly complete (pentaglucamine dimer peak at approximately 11.5 min)—see Figure 4. The remaining unannotated peaks were obtained from the sample matrix. Based on the equilibrium chemical equation, the added TCEP was primarily converted to TCEP oxide, and the residual TCEP was then inhibited by air oxidation back to the dimer before being added to the conjugation reaction. For simplicity, the glucosamine pentasaccharide can be added based on the input dimer, assuming >95% conversion to monomer under these conditions.

[0187] The identity of the pentadimer was confirmed using a 500 MHz instrument. 1 H and 13 The NMR spectra were determined by C NMR. A reference solution of t-butanol was prepared at 25 mg / mL in DO. Samples were prepared at 13 mg / mL in DO, and the reference solution was added to the sample. The composition of the final test sample was 10 mg / mL pentadimer and 5 mg / mL t-butanol. 1 H and 13 C spectra were acquired and integrated, and the resulting chemical shifts were assigned by comparison with theoretical shifts. 1 H NMR and 13 The C NMR spectra are shown in Figures 1 and 2, respectively. [Example 5]

[0188] Conversion of the pentasaccharide monomer of Example 4 with the TT-linker of Example 2 to provide the vaccine of the present invention (compound 18) The TT monomer-linker intermediate from Example 2 was reacted with increasing concentrations of 4 to 70 pentameric glucosamine molar equivalents (2 to 35 pentasaccharide dimer molar equivalents) for 4 hours at ambient temperature. Crude conjugates from each titration point were purified by partitioning through a 30 kDa MWCO membrane. Each purified conjugate sample was analyzed for protein content, payload density by SEC-MALS, and monomer / aggregate content by SEC-HPLC. The data indicated saturation of payload density at ≥50 pentameric glucosamine equivalents. Based on SEC-HPLC analysis, aggregate content increased with increasing pentasaccharide monomer charge, appearing to reach a steady-state level of approximately 4% increase starting at 30 pentameric glucosamine equivalents. Based on these results, the pentasaccharide dimer charge selected for subsequent conjugation reactions was 25 molar equivalents, corresponding to 50 molar equivalents of the theoretical charge of pentameric glucosamine.

[0189] A series of three test syntheses followed by a GMP synthesis of compound 18 were prepared as described above. The potency (by ELISA assay) and payload density (pentameric glucosamine to tetanus toxoid molar ratio) of each resulting product were evaluated. The results are shown in the table below.

[0190] [Table 2]

[0191] These results demonstrate the extremely high loading capacity of the compounds of the present invention. The foregoing description is provided merely to illustrate the present invention and is not intended to be limiting. Since modifications of the described embodiments incorporating the spirit and substance of the present invention may occur to those skilled in the art, the present invention should be broadly construed to include all modifications within the scope of the claims and their equivalents. [Example 6]

[0192] Tetanus toxoid purification by size exclusion chromatography. Aliquots of concentrated tetanus toxoid were purified by chromatography on a GE Healthcare 2.6 x 60 cm Superdex 200 column eluted with 10 mM NaHCO3 / 150 mM NaCl (pH 9.0) at 2.0 mL / min. Individual fractions (4.0 mL) were pooled based on analytical SEC-HPLC testing. Based on the purity of the TT monomer preparation obtained under these conditions (2.0 mL sample / 0.6% bed volume), the Superdex 200 loading was successfully increased by two-fold (4.0 mL sample / 1.2% bed volume) without significantly altering resolution. This change effectively reduced the number of chromatography cycles required for purification. SEC pools containing TT monomer of the desired quality were concentrated and buffer-exchanged using Amicon Ultra-15 Ultracel 30 kDa regenerated cellulose centrifugal filters. The purified TT was buffer-exchanged into 50 mM HEPES (pH 8.0) buffer for conjugation studies.

Claims

1. 1. A vaccine composition comprising a pharmaceutically acceptable excipient and an effective amount of a vaccine comprising at least 10, preferably about 10 to about 40, oligomeric β-(1→6)-glucosamine group units linked via a linker to a tetanus toxoid carrier, said oligomers comprising 3 to 12 repeating β-(1→6)-glucosamine units, with the proviso that less than about 40-fold percent of the total number of such units are N-acetylated; the vaccine composition contains less than about 3 percent detectable impurities having a molecular weight of less than about 100,000; Additionally, the composition comprises monomeric and dimeric toxoids with less than 5 percent detectable higher order oligomers; Still further, the vaccine composition, wherein the composition is maintained at a temperature sufficient to inhibit oligomerization of the toxoid in the vaccine without inducing denaturation.

2. 1. A vaccine composition comprising a pharmaceutically acceptable excipient and an effective amount of a vaccine comprising at least 25, preferably about 30 to about 40, oligomeric β-(1→6)-glucosamine group units linked via a linker to a tetanus toxoid carrier, wherein the oligosaccharide group comprises 3 to 12 repeating β-(1→6)-glucosamine units, with the proviso that less than about 40 percent of the total number of such units are N-acetylated; the vaccine composition contains less than 3 percent of detectable impurities each having a molecular weight of less than 50,000; Additionally, the composition comprises monomeric and dimeric toxoids with less than 5 percent detectable higher order oligomers; Still further, the vaccine composition wherein the composition is maintained at a temperature sufficient to inhibit oligomerization of the toxoid without inducing denaturation.

3. a pharmaceutically acceptable excipient and an effective amount of a compound of Formula I: (A-B) ｘ -C I wherein A is the following formula: 【Chemistry 1】 or mixtures thereof, and B comprises 3 to 12 repeating β-(1→6)-glucosamine units having the formula: 【Chemistry 2】 and The left side of the formula is attached to C and the right side is attached to A; C is tetanus toxoid; x is an integer from about 10 to about 40; y is an integer from 1 to 10; R is hydrogen or acetyl, provided that no more than 40% of said R groups are acetyl.

1. A vaccine composition comprising a vaccine compound of the composition contains less than 3 percent of detectable impurities having a molecular weight of less than about 100,000, said weight percent being based on the weight of the vaccine compound; Additionally, the composition comprises monomeric and dimeric toxoids with less than about 5 percent detectable higher order oligomers; Still further, the vaccine composition wherein the composition is maintained at a temperature sufficient to inhibit oligomerization of the toxoid without inducing denaturation.

4. The compound of formula I may be of formula II: (A' - B) ｘ - C II (Wherein A' is the following formula: 【Transformation 3】 where B, C, and x are as defined above. The vaccine composition of claim 3, represented by:

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