U.S. patent application number 11/818804 was filed with the patent office on 2008-05-08 for glucan preparations.
Invention is credited to Donald J. Cox, Ren-der Yang.
Application Number | 20080108114 11/818804 |
Document ID | / |
Family ID | 38832560 |
Filed Date | 2008-05-08 |
United States Patent
Application |
20080108114 |
Kind Code |
A1 |
Cox; Donald J. ; et
al. |
May 8, 2008 |
Glucan preparations
Abstract
A method for producing particulate .beta.-glucan utilizes
surfactant extraction to reduce lipid impurities that decompose to
volatile organic compounds (VOCs). The resulting particulate
.beta.-glucan is essentially free of VOCs.
Inventors: |
Cox; Donald J.; (Maple
Grove, MN) ; Yang; Ren-der; (Shrewsbury, MA) |
Correspondence
Address: |
BIOPOLYMER ENGINEERING , INC. DBA BIOTHERA , INC
C/O INTERLLEVATE , LLC
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Family ID: |
38832560 |
Appl. No.: |
11/818804 |
Filed: |
June 15, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60813971 |
Jun 15, 2006 |
|
|
|
Current U.S.
Class: |
435/101 ;
536/123.12 |
Current CPC
Class: |
C08L 5/00 20130101; C08B
37/0024 20130101; A61K 31/716 20130101; A61P 35/00 20180101; A61K
2039/545 20130101; A61P 37/04 20180101 |
Class at
Publication: |
435/101 ;
536/123.12 |
International
Class: |
C07H 1/00 20060101
C07H001/00; C12P 19/04 20060101 C12P019/04 |
Claims
1. A process for producing particulate .beta.-glucan having
immunostimulating properties, the process comprising: treating a
suspension of yeast cell walls with an alkaline solution; treating
the suspension of yeast cell walls with a surfactant; and
substantially purifying the particulate b-glucan.
2. The process of claim 1 wherein the alkaline solution and
surfactant are combined.
3. The process of claim 1 and further comprising: treating the
suspension of yeast cells with the alkaline Solution multiple
times.
4. The process of claim 1 and further comprising: treating the
suspension of yeast cells with the surfactant multiple times.
5. The process of claim 1 wherein the alkaline solution is sodium
hydroxide.
6. The process of claim 1 wherein the surfactant is one of
octylthioglucoside, Lubrol PX, Triton X-100, sodium lauryl sulfate,
sodium dodecyl sulfate, Nonidet P-40, Tween 20, ionic (anionic,
cationic, amphoteric) surfactants such as alkyl sulfonlates and
benzalkonium chlorides, nonionic surfactants such as
polyoxyethylene hydrogenated castor oils, polyoxyethylene sorbitol
fatty acid esters, polyoxyethylene sorbitan fatty acid esters,
polyoxyethylene glycerol fatty acid esters, polyethylene glycol
fatty acid esters and polyoxyethylene alkyl phenyl ethers or any
combination thereof.
7. A composition comprising particulate b-glucan having
substantially no volatile organic compounds.
8. A composition comprising particulate b-glucan having
substantially no lipids.
Description
[0001] This application claims the benefit of U.S. Ser. No.
60/813,971 entitled GLUCAN PREPARTIONS, filed on Jun. 15, 2006.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to compositions that include
.beta.-glucan. More particularly, the present invention relates to
soluble .beta.-glucan compositions and their use in stem cell
mobilization.
[0003] 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 .beta.(1-3)-linked 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 progenitor cell (HSPC)
mobilization.
[0004] 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 hemorrhage. As a result, patients
who undergo cytoreductive therapy must also receive therapy to
reconstitute bone marrow function (hematopoiesis).
[0005] 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 .beta.-glucan is a
soluble yeast-derived polysaccharide and has been shown previously
to induce hematopoietic stem and progenitor cell (HSPC)
mobilization.
SUMMARY OF THE INVENTION
[0006] In the present invention, yeast is cultured, harvested and
purified to yield particulate .beta.-glucan essentially free of
contaminating volatile organic compounds (VOCs). Particulate
.beta.-glucan is prepared by subjecting yeast cells or fragments
thereof to a series of alkaline, surfactant, and acidic extractions
that remove host cell impurities.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 is a schematic representation of a process for
producing particulate .beta.-glucan.
[0008] FIG. 2 is a schematic representation of a process for
producing soluble .beta.-glucan.
DETAILED DESCRIPTION OF THE INVENTION
Particulate .beta.-Glucan
[0009] FIG. 1 is an overview of method 10, which includes steps
12-22, for producing insoluble, or particulate, .beta.-glucan from
yeast. In step 12, a yeast culture is grown, typically, 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. fermentati, S.
rouxii, Schizosaccharomyces pombe, Kluyveromyces (K.) lactis, K.
fragilis, K. polysporus, Candida (C.) albicans, C. cloacae, C.
tropicalis, C. utilis, Hansenula (H.) wingei, H. arni, H. henricii,
H. americana, H. canadiensis, H. capsulata, H. polymorpha, Pichia
(P.) kluyveri, P. pastoris, P. polymorpha, P. rhodanesis, P.
ohmeri, Torulopsis (T.) bovina and T. glabrata.
[0010] 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 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 HCl and thymine HCl 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 about 0.02%.
[0011] 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 of
fermentation, samples are taken to measure the optical density, the
dry weight, and the microbial purity.
[0012] 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 .beta.-glucan that is desired,
steps to disrupt or fragment the yeast cells may be carried out.
Any known chemical, enzymatic or mechanical methods, or any
combination thereof may be used to carry out disruption or
fragmentation of the yeast cells.
[0013] At step 14, the yeast cells containing the .beta.-glucan are
harvested. When producing bulk .beta.-glucan, yeast cells are
typically harvested using continuous-flow centrifugation.
[0014] 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,
sodium dodecyl sulfate, Nonidet P-40, Tween 20 and the like. Ionic
(anionic, cationic, amphoteric) surfactants (e.g., alkyl
sulfonates, benzalkonium 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 be extracted one or
more times.
[0015] Extractions are usually carried out at temperatures between
about 70.degree. C. and about 90.degree. 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.
[0016] After each extraction, the solid phase containing the
.beta.-glucan is collected using 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 .beta.-glucan remains in the insoluble
cell wall material.
[0017] 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.degree. C. for approximately 60 minutes. 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.degree. 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 0.5%
Triton-X 100 and heated to 75.degree. C. for approximately 60
minutes.
[0018] 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. .beta.-glucan product.
[0019] Previous purification procedures resulted in .beta.-glucan
containing minute amounts of 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 free of
any VOCs.
[0020] 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
references: [0021] Blagovic, B., J. Rpcuc, M. Meraric, K. Georgia
and V. Maric. 2001. Lipid composition of brewer's yeast. Food
Technol. Biotechnol. 39:175-181. [0022] Shulze. 1995. Anaerobic
physiology of Saccharomyces cerevisiae. Ph.D. Thesis, Technical
University of Denmark.
[0023] Van Der Rest, M. 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. TABLE-US-00001 TABLE 1 Blagovic et al
(2001) Van Der Shulze (anaerobic Rest et al Fatty acid (1995)
growth) (1995) 10:0 Capric acid 1.1% 12:0 and 12:1 4.8% 7.3% Lauric
acid 14:0 and 14:1 8.8% 5.1% 7.0*% Myristic acid 16:0 Palmitic acid
26.8% 44.2% 12.8% 16:1 16.6% 16.9% 32.3% Palmitoleic acid 18:0
Stearic acid 6.1% 13.9% 8.0% 18.1 Oleic acid 25.7% 7.3% 28.0% 18:2
and higher 10.1% 5.3% 9.4% Linoleic acid, arachadonic acid and
others *includes lipids 10:0 to 14:1
[0024] 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 .beta.-glucan, some,
but not all fat was removed by centrifugation and wash steps. Thus,
a typical preparation might yield a fat content of 3-7%.
[0025] 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 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
.beta.-glucan.
[0026] The alkali wash steps commonly used in production of yeast
.beta.-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 acids that quickly decompose to various oxidative
products of fat decomposition.
[0027] 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 in the decomposition of a lipid, also known as a
triglyceride, is the liberation of the free fatty acid from the
triglyceride. Free fatty acids are virtually nonexistent in the
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. .COPYRGT. 1985. Editor: Owen R.
Fennema. Marcel Dekker, Inc.; DeMan, J. M. Chapter 2. Lipids In:
Principles of Food Chemistry C) 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
.beta.-glucan it is the well-known process of saponification that
is releasing unsaturated fatty acids that decompose as described
below.
[0028] The process of fat oxidation has several mechanisms. The
most common mechanism is autoxidation. The process is initiated by
the removal of a hydrogen from an olefenic compound to create a
free radical. The removal of hydrogen takes place at the 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.fwdarw.R*+H* (creation of a free
radical electron) 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. R*+O.sub.2.fwdarw.RO.sub.2* (formation of reactive
oxygenated free radical) RO.sub.2*+RH+ROOH+R* (ROOH is the reactive
hydroperoxide that decomposes to secondary reaction products such
as VOCs) The chain reaction continues until it is terminated by
free radicals combining with themselves to yield nonreactive
products. R*+R*.fwdarw.R--R R*+RO.sub.2*RO.sub.2R 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
.beta.-glucan preparations that produce the same end products.
ROOH.fwdarw.RO*+OH-- RO*.fwdarw.cleavage reactions form aldehydes,
alkyl radicals (which form hydrocarbons and alcohols), esters,
alcohols, and hydrocarbons.
EXAMPLE
[0029] ##STR1## Decomposition of the epoxide produces several
products including .fwdarw.C--C--C--C--C--C
(hexane)+O.dbd.C--C.dbd.C--C.dbd.O (2-butene-1,4 dial) Similarly,
if the epoxide forms between the 2 and 3 carbon bonds the chemistry
leads to: ##STR2##
[0030] In a similar manner, the formation of any VOCs identified in
.beta.-glucan 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 .beta.-glucan preparations as possible
creates a product that is more pure not only in terms of fat but
also in terms of VOC contamination.
[0031] 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.degree.
C. and 100.degree. C. for approximately 30 minutes to about 12
hours.
[0032] 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 HCl. The material is mixed with enough glacial acetic
acid to make a 0.1 M acetic acid solution, which is heated to
90.degree. C. for approximately 5 hours.
[0033] 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 .beta.-glucan product is then
collected, as shown by step 22. Again, collection is typically
carried out by centrifugation or continuous-flow
centrifugation.
[0034] At this point, a purified, particulate .beta.-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 .beta.-glucan particles
that have substantially retained in vivo morphology (whole glucan
particles) down to submicron-size particles.
[0035] As is well known in the art, particulate .beta.-glucan is
useful in many food, supplement and pharmaceutical applications.
Alternatively, particulate .beta.-glucan can be processed further
to form aqueous, soluble .beta.-glucan.
Soluble .beta.-Glucan
[0036] FIG. 2 is an overview of method 24, which includes steps
26-32, for producing aqueous, soluble .beta.-glucan. The starting
material used in method 24 is particulate .beta.-glucan, which may
be produced by method 10 or produced by any of a number of
previously used methods. The particulate .beta.-glucan starting
material may range in size from whole glucan particles down to
submicron-sized particles.
[0037] In step 26, particulate .beta.-glucan undergoes an acidic
treatment under pressure and elevated temperature to produce
soluble .beta.-glucan. Pelleted, particulate .beta.-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 mixture contains
about 0.61 g sodium acetate, 5.24 ml glacial acetic acid and 430 g
pelleted, particulate .beta.-glucan. The vessel is purged with
nitrogen to remove oxygen and increase the pressure within the
reaction vessel.
[0038] In a particular embodiment, the pressure inside the vessel
is brought to 35 PSI, and the suspension is heated to about
135.degree. C. for between about 4.5 and 5.5 hours. It was found
that under these conditions the .beta.-glucan will solubilize. As
the temperature decreases from 135.degree. C., the amount of
solubilization also decreases.
[0039] 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.
[0040] The increased pressure and temperature imparts advantages
over prior art processes for solubilizing .beta.-glucan by
virtually eliminating the use of hazardous chemicals from the
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.
[0041] 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 .beta.-glucan is solubilized, the
mixture is cooled to stop the reaction.
[0042] The crude, solubilized .beta.-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 .beta.-glucan. Other means known in the art may
also be used if desired. At step 28, the soluble .beta.-glucan is
clarified. Suitable clarification means include, for example,
centrifugation or continuous-flow centrifugation.
[0043] Next, the soluble .beta.-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 .mu.m filter.
[0044] Step 32 utilizes chromatography for further purification.
The soluble .beta.-glucan may 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 (HIC), the soluble .beta.-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.
[0045] In one example of HIC, a column is packed with Tosah
Toyopearl Butyl 650M resin (or equivalent). The column is packed
and qualified according to the manufacturer's recommendations.
[0046] Prior to loading, the column equilibration flow-through is
sampled for pH, conductivity and endotoxin analyses. The soluble
.beta.-glucan, conditioned in the higher concentration ammonium
sulphate solution, is loaded and then washed. The nature of the
soluble .beta.-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
.beta.-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 ensure that the hexose load does not
exceed the capacity of the resin. Fractions are collected and
analyzed for the soluble .beta.-glucan product.
[0047] 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 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.
[0048] The eluate is collected in fractions, and various
combinations of samples from the 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 .beta.-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 equal to 17%. The selected combination
of fractions is subsequently pooled.
[0049] At this point, the soluble .beta.-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.
[0050] The purification techniques described above result in an
improved soluble .beta.-glucan that provides specific advantages as
a pharmaceutical agent, which is discussed below. The soluble
.beta.-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 NMR determined that the product consists of
polymeric chains associated into triple helices.
[0051] The soluble .beta.-glucan is typically uncharged and
therefore has no pK.sub.a. It is soluble in water independent of
pH, and the viscosity increases as the concentration increases.
[0052] Table 2 summarizes the typical levels of impurities in a
soluble .beta.-glucan product utilizing whole glucan particles
produced by method 10. TABLE-US-00002 TABLE 2 Range of Levels
Observed Impurity Specification in 3 Batches HMW (>380 kD)
.ltoreq.10% 4-8% LMW (<25 kD) .ltoreq.17% 8-13% Reducing sugar
0.7-1.6% of total hexose* 1.0-1.1% of total hexose Glycogen
.ltoreq.10% of total hexose <5% of total hexose Mannan (as
mannose) .ltoreq.1% of recovered hexose <0.6 to .ltoreq.0.8% of
recovered hexose Chitin (as glucosamine) .ltoreq.2% of total hexose
0.2-0.5% of total hexose Protein .ltoreq.0.2% of total hexose
<0.2% of total hexose Yeast protein Characterization** <2
ng/mg hexose DNA Characterization <6.5 to <50 pg/mg hexose
Ergosterol Characterization <10 to <25 .mu.g/mg hexose Triton
X-100 Characterization <1 to <5 .mu.g/mg hexose Antifoam 204
Characterization <10 .mu.g/mg hexose *Total hexose is determined
by a colorimetric assay. Sugar polymers are hydrolyzed in sulphuric
acid and anthrone to form furfurals. The furfurals conjugate with
the anthrone to 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 .beta.-glucan falls
between those molecular weight ranges.
[0053] 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.
[0054] Because a new reducing end is generated with each chain
cleavage, reducing sugar is a monitor of chain stability. Reducing
sugars can be measured by the bicinchoninic acid (BCA) assay, which
is well known in the art.
[0055] Potential process impurities include other yeast cell
constituents such as DNA, yeast cell proteins, lipids and other
polysaccharides such as glycogen, miannan and chitin. DNA levels
can be analyzed using the slot hybridization assay (MDS PanLabs,
Seattle, Wash.). 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.
cerevisiae cell proteins (Cygnus Technology, Southport, N.C.).
Residual lipids may be monitored by evaluating ergosterol levels
using reversed-phase high-performance liquid chromatography
(RP-HPLC) with detection at 280 nm.
[0056] Glycogen is a polysaccharide comprised primarily of
.alpha.-1,4-linked glucose, and its 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.
[0057] Mannan is a branched polymer of .alpha.-1,6-linked mannose
with .alpha.-1,2- and .alpha.-1,3-branches that is monitored, as
mannose, by its monosaccharide composition. The product is added to
a reaction, and the mannose is hydrolyzed with trifluoroacetic acid
and analyzed by HPLC.
[0058] Chitin is a polymer of .beta.-1,4-N-acetyl glucosamine,
which is monitored by a calorimetric assay. Soluble O-glucan is
hydrolyzed with sulphuric acid, and the resulting glucosamine forms
a complex with Ehrlich's reagent that is measured calorimetrically.
These and other suitable assays are known to those skilled in the
art.
[0059] 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 selective ion monitoring with an electrospray mass
spectroscopy detector in positive mode.
[0060] Certain product specifications are proposed for utilizing
the soluble .beta.-glucan as a pharmaceutical agent. These
specifications are listed in Table 3. TABLE-US-00003 TABLE 3
Category Attribute Method Proposed Limits General Appearance Visual
Clear, colorless solution pH pH meter 5.0-7.5 Osmolality osmometer
260-312 mOsm Identity HR-GPC profile GPC-MALS Conforms to standard;
ratio of peak retention volumes: 0.8-1.2 Strength Concentration
(total Colorimetric hexose 0.85-1.15 mg/ml hexose) assay Impurities
HMW material GPC-MALS .ltoreq.10% LMW material GPC-MALS .ltoreq.17%
Reducing sugar BCA assay 0.7-1.6% of total hexose Residual protein
Colorimetric protein .ltoreq.0.2% of total assay hexose Chitin
(glucosamine) Colorimetric assay .ltoreq.2% of total hexose Mannan
(mannose) Monosaccharide .ltoreq.1% of recovered composition hexose
Glycogen Enzymatic .ltoreq.10% of total hexose Safety Endotoxin
PyroGene .ltoreq.0.25 EU*/ml recombinant Factor C assay Bioburden
Membrane filtration .ltoreq.5 CFU**/10 ml *colony forming unit
**endotoxin unit
[0061] As stated above, soluble .beta.-glucan produced by methods
10 and 24 is an improved product over prior art soluble
.beta.-glucan materials. Improvement is seen in clinical trial
results where soluble .beta.-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
.beta.-glucan. The results are shown in Table 4. TABLE-US-00004
TABLE 4 Related AE (occurring in .gtoreq.5% of Improved Soluble
total participants) Bf1.sup.1 .beta.-Glucan.sup.2 Body as a whole
Back pain 7% -- Fever 16% -- Headache 30% 8% Pain 7% --
Cardiovascular Vasodilation/Flushing -- 6% Digestive Nausea 7% 6%
Hemic/lymphatic Ecchymosis -- -- Leukocytosis -- -- Respiratory
Dyspnea -- 1% Musculoskeletal Athralgia 11% -- Skin/appendages
Urticaria 7% -- Rash -- -- Special senses Conjunctivitis 9% --
.sup.1maximum single dose 2.25 mg/kg .sup.2maximum single dose 6.0
mg/kg
[0062] Bfl is known by the tradename Betafectin.TM., a soluble
.beta.-glucan product developed by Alpha-Beta Technology, Inc. The
process to produce Betafectin.TM. utilized formic acid to
solubilize particulate .beta.-glucan material. In addition, Bf1 was
not subjected to any chromatography in its purification
process.
[0063] The studies were performed with a volunteer population of
healthy subjects. When compared to Bf1, study participants taking
the improved soluble .beta.-glucan reported fewer adverse events
even though the maximum dosage was more than 2.5 times that of Bf1.
Thus, a much higher dosage of the improved soluble .beta.-glucan
can be given at least without increasing, but likely actually even
decreasing, side effects. In addition, the improved soluble
.beta.-glucan does not induce biochemical mediators, such as
interleukin-1.beta. and tumor necrosis factor-.alpha., which cause
inflammatory side effects.
[0064] The processes of the present invention provide several
advantages over prior alt processes and result in improved O-glucan
products. The particulate .beta.-glucan is essentially free of
harmful VOCs. Solubilization of .beta.-glucan is safer and more
economical. In addition, solubilization of particulate
.beta.-glucan made by the present process results in soluble
.beta.-glucan with improved pharmaceutical qualities.
[0065] 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 encompassed by the appended claims.
* * * * *