AU2014201427B2 - Glucan preparations - Google Patents

Abstract

Particulate P-glucan is solubilized at elevated pressure and temperature to form soluble p-glucan. The method is safe and economical and produces a product that is an 5 improved pharmaceutical agent.

Description

2014201427 12 Mar 2014

GLUCAN PREPARATIONS

BACKGROUND OF THE INVENTION 5 This application is a divisional application of Australian patent application 2007258191, the entire disclosure of which is incorporated herein by way of this reference.

The present invention relates to compositions that include β-glucan. More particularly, the present invention relates to soluble β-glucan compositions and their use in 10 stem cell mobilization.

Glucans are generally described as polymers of glucose and are derived from yeast, bacteria, fungi and plants such as oats and barley. Glucans containing a β(1-3)-1ϊη1^ glucopyranose backbone are known to have biological activity, specifically they have been shown to modulate the immune system and more recently to induce hematopoietic stem and 15 progenitor cell (HSPC) mobilization.

Treatment of various cancers increasingly involves cytoreductive therapy, including high dose chemotherapy or radiation. These therapies decrease a patient’s white blood cell counts, suppress bone marrow hematopoietic activity, and increase their risk of infection and/or haemorrhage. As a result, patients who undergo cytoreductive therapy must also 20 receive therapy to reconstitute bone marrow function (hematopoiesis).

Despite advances in stem cell mobilization and techniques, up to 20-25% of patients exhibit poor mobilization and are not able to proceed with auto transplantation. PGG β-glucan is a soluble yeast-derived polysaccharide and has been shown previously to induce hematopoietic stem and progenitor cell (HSPC) mobilization. 25 The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. 30 Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises”, is not intended to exclude other additives, components, integers or steps 35 1 2014201427 15 Aug 2016

SUMMARY OF THE INVENTION

In one aspect the invention provides a composition comprising underivatized, soluble β-glucan having an average molecular weight between about 120,000 Da and about 205,000 Da and immunostimulatory properties, wherein the β-glucan does not induce 5 biochemical mediators which cause inflammatory side effects, and can be administered in single doses up to about 6 mg/kg, and wherein the underivatized, soluble β-glucan is produced by a process comprising applying about 35 PSI of pressure to a suspension of particulate β-glucan and acid, and heating the suspension to about 135°C for a time sufficient to form the underivatized, soluble β-glucan. 10 In the present invention, yeast is cultured, harvested and purified to yield particulate β-glucan essentially free of contaminating volatile organic compounds (VOCs). Particulate β-glucan is prepared by subjecting yeast cells or fragments thereof to a series of alkaline, surfactant, and acidic extractions that remove host cell impurities.

Particulate β-glucan, produced by the above process or by prior art methods, is 15 solubilized in an acidic solution at elevated temperature and pressure. The resulting soluble β-glucan is then clarified and purified using hydrophobic interaction chromatography (HIC) followed by gel-permeating chromatography (GPC). As a pharmaceutical agent, the soluble β-glucan can be administered at higher doses without increasing, or in fact decreasing, observed side effects or adverse events. la 2014201427 12 Mar 2014 5 BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic representation of a process for producing particulate β- glucan.

FIG. 2 is a schematic representation of a process for producing soluble β-glucan. DETAILED DESCRIPTION OF THE INVENTION

Particulate β-glucan

Fig. 1 is an overview of method 10, which includes steps 12-22, for producing insoluble, or particulate, β-glucan from yeast. In step 12, a yeast culture is grown, typically, 10 in a shake flask or fermenter. The yeast strain utilized for the present invention can be any strain, examples of which include Saccharomyces (S.) cerevisiae, S. delbrueckii, S, rosei, S. microellipsodes, S. carlsbergensis, S. bisporus, S. fermented, S. rouxii, Schizosaccharomyces pombe, Kluyveromyces (K.) lactis, K. fragilis, K. polysporus, Candida (C.) albicans, C. cloacae, C. tropkalis, C. utilis, Hansenula (H.) wingei, H. arni, 15 H. henricii, H. americana, H. canadiensis, H. capsulata, H. potymorpha, Pichia (P.) kluyveri, P. pastoris, P. polymorpha, P. rhodanesis, P. ohmeri, Torulopsis (T.) bovina and T. glabrata.

In one embodiment of bulk production, a culture of yeast is started and expanded stepwise through a shake flask culture into a 250-L scale production fermenter. The yeast 20 are grown in a glucose-ammonium sulfate medium enriched with vitamins, such as folic acid, inositol, nicotinic acid, pantothenic acid (calcium and sodium salt), pyridoxine HC1 and thymine HC1 and trace metals from compounds such as ferric chloride, hexahydrate; zinc chloride; calcium chloride, dihydrate; molybdic acid; cupric sulfate, pentahydrate and boric acid. An antifoaming agent such as Antifoam 204 may also be added at a concentration of 25 about 0.02%.

The production culture is maintained under glucose limitation in a fed batch mode. During seed fermentation, samples are taken periodically to measure the optical density of the culture before inoculating the production fermenter. During production fermentation, samples are also taken periodically to measure the optical density of the culture. At the end 30 of fermentation, samples are taken to measure the optical density, the dry weight, and the microbial purity.

If desired, fermentation may be terminated by raising the pH of the culture to at least 11.5 or by centrifuging the culture to separate the cells from the growth medium. In addition, depending on the size and form of purified β-glucan that is desired, steps to 35 disrupt or fragment the yeast cells may be carried out. Any known chemical, enzymatic or 2 2014201427 12 Mar 2014 mechanical methods, or any combination thereof may be used to carry out disruption or fragmentation of the yeast cells.

At step 14, the yeast cells containing the β-glucan are harvested. When producing bulk β-glucan, yeast cells are typically harvested using continuous-flow centrifugation. 5 Step 16 represents the initial extraction of the yeast cells utilizing one or more of an alkaline solution, a surfactant, or a combination thereof. A suitable alkaline solution is, for example, 0,1 M-5 M NaOH. Suitable surfactants include, for example, octylthioglucoside, Lubrol PX, Triton X-100, sodium lauryl sulfate (SDS), Nonidet P-40, Tween 20 and the like. Ionic (anionic, cationic, amphoteric) surfactants (e.g., alkyl sulfonates, benzalkonium 10 chlorides, and the like) and nonionic surfactants (e.g., polyoxyethylene hydrogenated castor oils, polyoxyethylene sorbitol fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene glycerol fatty acid esters, polyethylene glycol fatty acid esters, polyoxyethylene alkyl phenyl ethers, and the like) may also be used. The concentration of surfactant will vary and depend, in part, on which surfactant is used. Yeast cell material may 15 be extracted one or more times.

Extractions are usually carried out at temperatures between about 70°C and about 90°C. Depending on the temperature, the reagents used and their concentrations, the duration of each extraction is between about 30 minutes and about 3 hours.

After each extraction, the solid phase containing the β-glucan is collected using 20 centrifugation or continuous-flow centrifugation and resuspended for the subsequent step. The solubilized contaminants are removed in the liquid phase during the centrifugations, while the β-glucan remains in the insoluble cell wall material.

In one embodiment, four extractions are carried out. In the first extraction, harvested yeast cells are mixed with 1.0 M NaOH and heated to 90°C for approximately 60 minutes. 25 The second extraction is an alkaline/surfactant extraction whereby the insoluble material is resuspended in 0.1 M NaOH and about 0.5% to 0.6% Triton X-100 and heated to 90°C for approximately 120 minutes. The third extraction is similar to the second extraction except that the concentration of Triton X-100 is about 0.05%, and the duration is shortened to about 60 minutes. In the fourth extraction, the insoluble material is resuspended in about 30 0.5% Triton-X 100 and heated to 75°C for approximately 60 minutes.

The alkaline and/or surfactant extractions solubilize and remove some of the extraneous yeast cell materials. The alkaline solution hydrolyzes proteins, nucleic acids, mannans, and lipids. Surfactant enhances the removal of lipids and other hydrophobic impurities, which provides an additional advantage yielding an improved β-glucan product. 35 Previous purification procedures resulted in β-glucan containing minute amounts of 3 2014201427 12 Mar 2014 volatile organic compounds (VOCs). Previous studies have shown that VOCs are produced by the release of fat as free fatty acid, which quickly decomposes into various VOCs. In most cases, the amounts detected are not enough to cause harm, however, it is an obvious benefit to have a product that is administered to humans or other animals that is essentially 5 free of any VOCs.

Fat content of the yeast Saccharomyces cerevisiae produced by aerobic and anaerobic growth ranges from about 3% to about 8%. The fat content varies depending on growth conditions of the yeast. Table 1 provides an overview of the typical fat composition of the yeast Saccharomyces cerevisiae. The data is from the following 10 references: • Blagovic, B., J. Rpcuc, M. Meraric, K. Georgia and V. Marie. 2001. Lipid composition of brewer’s yeast. FoodTechnol. Biotechnol. 39:175*181. • Shulze. 1995. Anaerobic physiology of Saccharomyces cerevisiae. Ph.D. Thesis, Technical University of Denmark. 15 · Van Der Rest, Μ. E., A. H. Kamming, A Nakano, Y. Anrak, B. Poolman and W. N. Koning. 1995. The plasma membrane of Saccharomyces cerevisiae: structure, function and biogenesis. Microbiol. Rev. 59:304-322. 20 TABLE 1 Fatty acid Shulze (1995) Blagovic ct al (2001) (anaerobic growth) Van Der Rest et al (1995) 10:0 Capric acid 1.1% 12:0 and 12:1 Laurie acid 4.8% 7.3% 14:0 and 14:1 Myristic acid 8.8% 5.1% 7.0 Vo 16:0 Palmitic acid 26.8% 44.2% 12.8% 16:1 Palmitoleic acid 16.6% 16.9% 32.3% 18:0 Stearic acid 6.1% 13.9% 8.0% 18.1 Oleic acid 25.7% 7.3% 28.0% 18:2 and higher Linoleic acid, arachadonic acid and others 10.1% 5.3% 9.4% includes lipids 10:0 to 14:1 4 2014201427 12 Mar 2014

Yeast cell wall material typically contains 10-25% fat depending on yeast type and growth conditions. Presently, during processing of yeast cell wall material into β-glucan, some, but not all fat is removed by centrifugation and wash steps. Thus, a typical preparation might yield a fat content of 3-7%. 5 The manufacturing process typically involves steps to remove mannoproteins, lipids and other undesirable components of the yeast cell wall. Some key steps common to this processing are various wash steps that employ acid and alkali in separate washing steps to liberate certain cell wall components. Several of the steps use an alkali wash process where an alkali, usually sodium hydroxide, is added to the liquid cell wall 10 suspension. One of the purposes of the alkali is to remove lipid by forming the free fatty acids of the lipid. The result is a reduction in fat content of the β-glucan.

The alkali wash steps commonly used in production of yeast β-glucan leave behind residual fatty acids and partially degraded fat triglycerides that have increased reactivity. The direct result of the alkali wash process is the release of reactive free fatty 15 acids that quickly decompose to various oxidative products of fat decomposition.

Numerous researchers have detailed the fact that poly-unsaturated fats decompose during storage. Although a triglyceride can autoxidize in the presence of oxygen, it is more common for free fatty acids to undergo oxidative decomposition. The normal step iri the decomposition of a lipid, also known as a triglyceride, is the liberation of the free 20 fatty acid from the triglyceride. Free fatty acids are virtually nonexistent in die tissues of living organisms, but decomposition is common when the organism dies or is harvested for further processing such as occurs with oilseeds and in rendering of animal fat. (Nawar, W. W. Chapter 4. Lipids. In: Food Chemistry. © 1985. Editor: Owen R. Fennema. Marcel Dekker, Inc.; DeMan, J. M. Chapter 2. Lipids In: Principles of Food 25 Chemistry © 1985. AVI Publishing Co., Inc.) In many triglycerides, the 2-position of the glyceride molecule is occupied by an unsaturated fat. In the case of alkali treatment of β-glucan it is the well-known process of saponification that is releasing unsaturated fatty acids that decompose as described below.

The process of fat oxidation has several mechanisms. The most common 30 mechanism is autoxidation. The process is initiated by the removal of hydrogen from an olefenic compound to create a free radical. The removal of hydrogen takes place at the 5 2014201427 12 Mar 2014 carbon atom next to the double bond in the fat. The reaction is initiated by various free-radical generating factors such as UV light, metals, singlet oxygen, etc. RH -> R* + H‘ (creation of a free radical electron) 5

The second step is the addition of oxygen to cause formation of a peroxy-free radical, which propagates the chain reaction by extracting hydrogen from another unsaturated fatty acid. 10 R* + O2 ->R(Y (formation of reactive oxygenated free radical) R02· + RH -> ROOH + R' (ROOH is the reactive hydroperoxide that decomposes to secondary reaction products such as VOCs) 15 The chain reaction continues until it is terminated by free radicals combining with themselves to yield nonreactive products.

R* + R‘ -» R-R

20 R’ + RCV -» R02R

The following are the chemical reactions that occur to form the VOCs. Linoleic acid is used as a model for the chemistry, but there are other unsaturated fatty acids present in the yeast cell wall and in yeast β-glucan preparations that produce the same end 25 products. ROOH RO* + OH- RO* cleavage reactions form aldehydes, alkyl radicals (which form hydrocarbons and 30 alcohols), esters, alcohols, and hydrocarbons. 6 2014201427 12 Mar 2014

Example:

H

Ο H

ο I C-C-C=C-C=C-C-C-C-C-(C)7-COO -> C-C-C-C-C~C=C-C=C-C-0 (2,4 decadienal) 5 V(/

Η H

I I C-C-C-C-C-C=C-C=C-C-0.-» (epoxide formation) -> C-C-C-C-C-C-C-C=C-C-0 o 10

Decomposition of the epoxide produces several products including C-C-C-C-C-C (hexane) + 0=C-C=C-C=0 (2-butene-l,4 dial)

Similarly, if the epoxide forms between the 2 and 3 carbon bonds the chemistry leads to: 15

C-C-C-C-C-C=C-C^rC-0 (2,3 epoxide of 2,4-decadienal) -> ethanol and 2-octenal O

In a similar manner, the formation of any VOCs identified in β-glucan 20 preparations can be accounted for by the autoxidation reactions that occur with decomposition of the reactive species of peroxides formed during fatty acid oxidation. Therefore, the removal of as much fat from β-glucan preparations as possible creates a product that is more pure not only in terms of fat but also in terms of VOC contamination. 25 Referring back to Fig. 1, the next step in the purification process is an acidic extraction shown at step 18, which removes glycogen. One or more acidic extractions are accomplished by adjusting the pH of the alkaline/surfactant extracted material to between about 5 and 9 and mixing the material in about 0.05 M to about 1.0 M acetic acid at a temperature between about 70°C and 100°C for approximately 30 minutes to about 12 30 hours. 7 2014201427 12 Mar 2014

In one embodiment, the insoluble material remaining after centrifugation of the alkaline/surfactant extraction is resuspended in water, and the pH of the solution is adjusted to about 7 with concentrated HC1. The material is mixed with enough glacial acetic acid to make a 0.1 M acetic acid solution, which is heated to 90°C for 5 approximately 5 hours.

At step 20, the insoluble material is washed. In a typical wash step, the material is mixed in purified water at about room temperature for a minimum of about 20 minutes. The water wash is carried out two times. The purified β-glucan product is then collected, as shown by step 22. Again, collection is typically carried out by 10 centrifugation or continuous-flow centrifugation.

At this point, a purified, particulate β-glucan product is formed. The product may be in the form of whole glucan particles or any portion thereof, depending on the starting material. In addition, larger sized particles may be broke down into smaller particles. The range of product includes β-glucan particles that have substantially retained in vivo 15 morphology (whole glucan particles) down to submicron-size particles.

As is well known in the art, particulate β-glucan is useful in many food, supplement and pharmaceutical applications. Alternatively, particulate β-glucan can be processed further to form aqueous, soluble β-glucan. 20 Soluble β-glucan

Fig. 2 is an overview of method 24, which includes steps 26-32, for producing aqueous, soluble β-glucan. The starting material used in method 24 is particulate β-glucan, which may be produced by method 10 or produced by any of a number of previously used methods. The particuate β-glucan starting material may range in size from whole glucan 25 particles down to submicron-sized particles.

In step 26, particulate β-glucan undergoes an acidic treatment under pressure and elevated temperature to produce soluble β-glucan. Pelleted, particulate β-glucan is resuspended and mixed in a sealable reaction vessel in a buffer solution and brought to pH 3.6. Buffer reagents are added such that every liter, total volume, of the final suspension 30 mixture contains about 0.61 g sodium acetate, 5.24 ml glacial acetic acid and 430 g pelleted, particulate β-glucan. The vessel is purged with nitrogen to remove oxygen and increase the pressure within the reaction vessel. 8 2014201427 12 Mar 2014

In a particular embodiment, the pressure inside the vessel is brought to 35 PSI, and the suspension is heated to about 135°C for between about 4.5 and 5.5 hours. It was found that under these conditions the β-glucan will solubilize. As the temperature decreases from 135°C, the amount of solubilization also decreases. 5 It should be noted that this temperature and pressure are required in the embodiment just described. Optimization of temperatures and pressures may be required if any of the reaction conditions and/or reagents are altered.

The increased pressure and temperature imparts advantages over prior ait processes for solubilizing β-glucan by virtually eliminating the use of hazardous chemicals from the 10 process. Hazardous chemicals that have previously been used include, for example, flammable VOCs such as ether and ethanol, very strong acids such as formic acid and sulphuric acid and caustic solutions of very high pH. The present process is not only safer, but, by reducing the number of different chemicals used and the number of steps involved, is more economical. 15 The exact duration of heat treatment is typically determined experimentally by sampling reactor contents and performing gel permeation chromatography (GPC) analyses. The objective is to maximize the yield of soluble material that meets specifications for high resolution-GPC (HR-GPC) profile and impurity levels, which are discussed below. Once the β-glucan is solubilized, the mixture is cooled to stop the reaction. 20 The crude, solubilized β-glucan may be washed and utilized in some applications at this point, however, for pharmaceutical applications further purification is performed. Any combination of one or more of the following steps may be used to purify the soluble β-glucan. Other means known in the art may also be used if desired. At step 28, die soluble β-glucan is clarified. Suitable clarification means include, for example, centrifugation or 25 continuous-flow centrifugation.

Next, the soluble β-glucan may be filtered as shown by step 30. In one embodiment, the material is filtered, for example, through a depth filter followed by a 0.2 μιη filter.

Step 32 utilizes chromatography for further purification. The soluble β-glucan may 30 be conditioned at some point during step 28 or step 30 in preparation for chromatography. For example, if a chromatographic step includes hydrophobic interaction chromatography (HIQ, the soluble β-glucan can be conditioned to the appropriate conductivity and pH with a solution of ammonium sulphate and sodium acetate. A suitable solution is 3.0 M ammonium sulfate, 0.1 M sodium acetate, which is used to adjust the pH to 5.5.

35 In one example of HIC, a column is packed with Tosah Toyopearl Butyl 650M 9 2014201427 12 Mar 2014 resin (or equivalent). The column is packed and qualified according to the manufacturer’s recommendations.

Prior to loading, the column equilibration flow-through is sampled for pH, conductivity and endotoxin analyses. The soluble β-glucan, conditioned in the higher 5 concentration ammonium sulphate solution, is loaded and then washed. The nature of the soluble β-glucan is such that a majority of the product will bind to the HIC column. Low molecular weight products as well as some high molecular weight products are washed through. Soluble β-glucan remaining on the column is eluted with a buffer such as 0.2 M ammonium sulfate, 0.1 M sodium acetate, pH 5.5. Multiple cycles may be necessary to 10 ensure that the hexose load does not exceed the capacity of the resin. Fractions are collected and analyzed for the soluble β-glucan product.

Another chromatographic step that may be utilized is gel permeation chromatography (GPC). In one example of GPC, a Tosah Toyopearl HW55F resin, or equivalent is utilized and packed and qualified as recommended by the manufacturer. The 15 column is equilibrated and eluted using citrate-buffered saline (0.14 M sodium chloride, 0.011 M sodium citrate, pH 6.3). Prior to loading, column wash samples are taken for pH, conductivity and endotoxin analyses. Again, multiple chromatography cycles may be needed to ensure that the load does not exceed the capacity of the column.

The eluate is collected in fractions, and various combinations of samples from the 20 fractions are analyzed to determine the combination with the optimum profile. For example, sample combinations may be analyzed by HR-GPC to yield the combination having an optimized HR-GPC profile. In one optimized profile, the amount of high molecular weight (HMW) impurity, that is soluble β-glucans over 380,000 Da, is less than or equal to 10%. The amount of low molecular weight (LMW) impurity, under 25,000 Da, is less than or 25 equal to 17%. The selected combination of fractions is subsequently pooled.

At this point, the soluble β-glucan is purified and ready for use. Further filtration may be performed in order to sterilize the product. If desired, the hexose concentration of the product can be adjusted to about 1.0 ± 0.15 mg/ml with sterile citrate-buffered saline.

The purification techniques described above result in an improved soluble β-glucan 30 that provides specific advantages as a pharmaceutical agent, which are discussed below. The soluble β-glucan has an average molecular weight between about 120,000 Da and about 205,000 Da and a molecular weight distribution (polydispersity) of not more than 2.5 as determined by HR-GPC with multiple angle light scattering (HR-GPC/MALS) and differential refractive index detection. Powder X-ray diffraction and magic-angle spinning 35 NMR determined that the product consists of polymeric chains associated into triple 10 2014201427 12 Mar 2014 helices.

The soluble β-glucan is typically uncharged and therefore has no pKa. It is soluble in water independent of pH, and the viscosity increases as the concentration increases.

Table 2 summarizes the typical levels of impurities in a soluble β-glucan product 5 utilizing whole glucan particles produced by method 10. TABLE2

Impurity Specification Range of Levels Observed in 3 Batches HMW (>380 kD) £10% 4-8% LMW «25 kD) £17% gIT3^ Reducing sugar 0.7-1.6% of total hexose* 1.0-1.1% of total hexose Glycogen £10% of total hexose <5% of total hexose Mannan (as mannose) £l % of recovered hexose <0.6 to £0.8% of recovered hexose Chitin (as glucosamine) £2% of total hexose 0.2-0.5% of total hexose Protein £0.2% of total hexose <02% of total hexose Yeast protein Characterization* * <2 ng/mg hexose DNA Characterization <6.5 to <50 pg/mg hexose Ergosterol Characterization <10 to <25 pg/mg hexose Triton X-100 Characterization <1 to <5 pg/mg hexose Antifoam 204 Characterization <10 pg/mg hexose sulphuric acid and anthrone to form ftirfurals. The furfurals conjugate with the anthrone to 10 yield a chromophore, which is measured spectrophotometrically. **Limits were not specified.

Product-related impurities include material with molecular weights greater than 380,000 daltons or less than 25,000 daltons, because it has been found that the improved soluble β-15 glucan falls between those molecular weight ranges.

An additional measure of product-related impurities is reducing sugar. Each glucan polysaccharide chain ends in the aldehyde form (reducing sugar) of the sugar. Thus, the amount of reducing sugar serves as an indication of the number of chains in the preparation. Because a new reducing end is generated with each chain cleavage, reducing sugar, is a 20 monitor of chain stability. Reducing sugars can be measured by the bicinchoninic acid (BCA) assay, which is well known in the art.

Potential process impurities include other yeast cell constituents such as DNA, yeast cell proteins, lipids and other polysaccharides such as glycogen, mannan and chitiii. DNA levels can be analyzed using the slot hybridization assay (MDS PanLabs, Seattle,. WA). 25 Residual protein may be determined by a colorimetric assay, for protein or by a more sensitive commercial enzyme-linked immunosorbent assay (ELISA) that measures S. 11 2014201427 12 Mar 2014 cerevisiae cell proteins (Cygnus Technology, Southport, North Carolina). Residual lipids may be monitored by evaluating ergosterol levels using reversed-phase high-performance liquid chromatography (RP-HPLQ with detection at 280 nm.

Glycogen is a polysaccharide comprised primarily of a-l,4-linked glucose, and its 5 presence can be determined by an enzymatic assay. The product is added to an enzymatic reaction containing amyloglucosidase, which liberates glucose from glycogen, generating reducing sugars. The reducing sugars are measured by the BCA assay.

Mannan is a branched polymer of a-l,6-linked mannose with a-1,2- and a-13-branches that is monitored, as mannose, by its monosaccharide composition. The product is 10 added to a reaction, and the mannose is hydrolyzed with trifluoroacetic acid and analyzed by HPLC.

Chitin is a polymer of β-Ι,Φ-Ν-acetyl glucosamine, which is monitored by a colorimetric assay. Soluble β-glucan is hydrolyzed with sulphuric acid, and the resulting glucosamine forms a complex with Ehrlich’s reagent that is measured colorimetrically. 15 These and other suitable assays are known to those skilled in the art.

Potential non-yeast impurities originating from components added during the manufacturing process include Triton X-100 (surfactant) and Antifoam 204 (antifoaming agent). Reversed-phase HPLC (RP-HPLC) with detection at 280 nm can be used to discern any residual Triton X-100. Antifoam 204 is assessed by a RP- HPLC method using 20 selective ion monitoring with an electrospray mass spectroscopy detector in positive mode.

Certain product specifications are proposed for utilizing the soluble β-glucan as a pharmaceutical agent. These specifications are listed in Table 3. TABLE 3

Category

Attribute

Method

Proposed Limits

General

Appearance

Visual

Clear, colorless solution

Osmolality pH meter osmometer 5.0-7.5 260-312 mOsm

Identity HR-GPC profile

GPC-MALS

Conforms to standard; ratio of peak retention volumes; 0.8-1.2

Strength impurities

Concentration (total hexose) HMW material LMW material

Reducing sugar

Residual-protein

Colorimetric hexose

GPC-MALS GPC-MALS BCA assay

Colorimetric protein 12 0.85-1.15 mg/rnl *10% *17% 0.7-1.6% of total hexose -*0.2%-eftotal- assay hexose Chitin (glucosamine) Colorimetric assay £2% of total hexose Mannan (mannose) Monosaccharide composition £l% of recovered hexose Glycogen Enzymatic £10% of total hexose Safety Endotoxin PyroGene recombinant Factor C assay ^0.25 EU*/ml Bioburden Membrane filtration £5 CPU **/10 ml * colony forming unit * *endotoxin unit 2014201427 12 Mar 2014

As stated above, soluble β-glucan produced by methods 10 and 24 is an improved 5 product over prior art soluble β-glucan materials. Improvement is seen in clinical trial results where soluble β-glucan of the present invention given at a much higher maximum dose showed the same or fewer adverse events (AEs) as lower maximum doses of prior art soluble β-glucan. The results are shown in Table 4. 10 TABLE 4

Related AE- (occurring in a 5% of total participants) Bfl1 Improved Soluble β-Glucan2 Body as a whole Back pain Fever Headache Pain 7% 16% 30% 7% 8% Cardiovascular Vasodilation/Flushing WM 6% Digestive Nausea 7% 6% Hemic/Iymphatic Ecchymosis Leukocytosis — — Respiratory Dyspnea 1% Musculoskeletal Athralgia 11% Skin/appendages Urticaria Rash 7% - Special senses Conjunctivitis 9% ‘maximum single dose 2.25 mg/kg 2maximum single dose 6.0 mg/kg 13 2014201427 12 Mar 2014

Bfl is known by the tradename Betafectin™, a soluble β-glucan product developed by Alpha-Beta Technology, Inc. The process to produce Betafectin™ utilized formic acid to solubilize particulate β-glucan material. In addition, Bfl was not subjected to any chromatography in its purification process. 5 The studies were performed with a volunteer population of healthy subjects. When compared to Bfl, study participants taking the improved soluble β-glucan reported fewer adverse events even though the maximum dosage was more than 2.5 times that of Bfl. Thus, a much higher dosage of the improved soluble β-glucan can be given at least without increasing, but likely actually even decreasing, side effects. In addition, the improved 10 soluble β-glucan does not induce biochemical mediators, such as interleukin-1 β and tumor necrosis factor-α, which cause inflammatory side effects.

The processes of the present invention provide several advantages over prior art processes and result in improved β-glucan products. The particuate β-glucan is essentially free of harmful VOCs. Solubilization of β-glucan is safer and more economical. In 15 addition, solubilization of particulate β-glucan made by the present process results in soluble β-glucan with improved pharmaceutical qualities.

While this invention has been shown and described with references to particular embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention 20 encompassed by the appended claims. 14

Claims (7)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS

1. A composition comprising underivatized, soluble β-glucan having an average molecular weight between about 120,000 Da and about 205,000 Da and immunostimulatory properties, wherein the β-glucan does not induce biochemical mediators which cause inflammatory side effects, and can be administered in single doses up to about 6 mg/kg, and wherein the underivatized, soluble β-glucan is produced by a process comprising applying about 35 PSI of pressure to a suspension of particulate β-glucan and acid, and heating the suspension to about 135°C for a time sufficient to form the underivatized, soluble β-glucan.

2. The composition of claim 1, wherein the biochemical mediators are interleukin-1 β, tumor necrosis factor-α, or both.

3. The composition of claim 1 or claim 2, wherein the underivatized, soluble β-glucan is derived from Saccharomyces cerevisiae.

4. The composition of any one of claims 1 to 3, wherein the underivatized, soluble β-glucan contains no more than about 1.6% reducing sugar of total hexose.

5. The composition of any one of claims 1 to 4, wherein the underivatized, soluble β-glucan contains less than about 25 pg/'mg of hexose.

6. The composition of any one of claims 1 to 5, wherein no more than 8% of humans administered the undervatized, soluble β-glucan show any one adverse event.

7. The composition of any one of claims 1 to 6, wherein the composition comprises underivatized soluble β-glucan having a molecular weight greater than 380,000 Da in an amount from 4% to 8% and underivatized soluble β-glucan having a molecular weight less than 25,000 Da in an amount from 8% to 13%.