Note: Descriptions are shown in the official language in which they were submitted.
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- 1 -
PRQCESS FOR ISOLATING IMl\~UNOGT,OBULINS IN WHEY
Field of the Invention
The present invention is directed to a process of isolating imml1noglobulins from whey
s and whey concentrate, and a concentrated immlm~globulin product which is highly purified and
~ readily ~lministered. The present invention is also directed to a precess for producing and
selecting antigens for a (bovine) vaccine, a method of treating infection caused by enterotoxic
E. coli (ETEC) and immlln~globulin products effective for tre~Jment of ETEC infection.
Back~round of the Invention
Tmmllnnglobulins or antibodies are made by higher ~nim~l~ in response to the presence of
a foreign composition. Such a foreign composition, capable of eliciting an immunc response, is
referred to as an antigen. Tmmlmnglobulins are complex proteins which are capable of
specifically binding or ~tt~- hing to the antigen.
Tmmlm~globulins play an important role in a host organism's fight against disease.
5 Tmmlmoglobulins, often abbreviated as Ig, or antibodies abbreviated Ab, are made in several
dirr~rt;ll~ forms. These classes of immunoglobulin are IgG, which is abundant in intern~l body
fluids and certain lacteal secretions; IgA, abundant in sero-mucous secretions; IgM, an effective
aggluLi~ ol, IgD, found on the surface of lymphocytes; and IgE, involved in allergic responses.
IgG is the principle immunoglobulin in bovine milk and colostrum, while IgA is the dominant
20 immllnoglobulin in lacteal secretions in hllm~ns The level of antigen specific immllnc-globulins
present in milk or colostrum can be increased through parenteral or intra m~mm~ry
""~",l,i7~tion regimes.
Hyperimmune imml-noglobulins derived from bovine milk or colostrum have been
proposed for use in a variety of ph~rm~- eutical/medicinal applications. Among these are oral
25 and topical applications for the tre~tment or ~ven~ion of infections diseases caused by
pathogens including C. parvum, rotavirus, H. pylori, E. coli, Shigella species, S. mutans and
Candida species. Enterotoxigenic E. coli (ETEC) causes the disease associated with Traveler's
r1i~rrhe~
Immunoglobulins for this purpose can be from colostrum, which is the first 4-5 milkin~s
30 after calving, or from milk produced during the rem~inc1er of the lactation. While
immunoglobulins are present in relatively high concentrations (20-100 mg/ml) in colostrum
compared to milk (0.3-0.5 mg/ml), production of commercial quantities of immunoglobulins
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from colostrum is made difficult both by limited supplies and the complexities of collecting and
proce.ssin~ small volumes from individual cows on a commercial scale.
Milk in contrast, is in abundant supply and has well established systems for collection
and processing. While immunoglobulin levels in milk are low, it is well kno~,vn that the majority
s of milk immunoglobulins pass into whey during conventional cheese making. Whey is a low
cost and abundant byproduct of the cheese making industry and is readily available as a raw
material for Ig purification.
In addition to the relatively low concentrations of immunoglobulins in milk and whey,
the other difficulty in producing commercial quantities of purified whey immunoglobulins is the
lo presence of high concenkations (4-6 mg/ml) of non-immunoglobulin proteins including ~-
lactoglobulin and cc-lactalbumin. Removal of greater than 90% of these proteins is required to
produce a final product in which immunnglobulins constitute greater than 60% of the total
protein.
Immunoglobulin products have been proposed for the keatment of ETEC in humsln~
However, these products have been unsuccessful due to the large volume, mass or cost of an
effective dose. The large volume and mass of an effective dose typically limits ~rlmini~tration to
subjects in the form of a food bar, recon~tit~lt~l milk-like product or the like. The most desirable
dose form would be a small tablet or capsule which affords portability and convenience. Such a
forrn~ tion requires a highly con~ d~ed immunoglobulin preparation of high purity. Previous
efforts to develop immunoglobulin-based anti-ETEC products have failed to solve the need for a
portable and shelf stable dose form having high specific activity against ETEC.
Production of commercial quantities of immunoglobulins from whey, therefore, requires
processing methods which allow for convenient and low cost removal of non-immunoglobulin
proteins and high-throughput of large liquid volumes.
Summ~ry of the Invention
The present invention features a method for purifying immunoglobulins from whey,whey concentrate, whey fractions or partially deproteinized whey concentrate which provides a
final whey protein L~rep~lion that is greater than 60% by weight immunoglobulins. The method
comprises forrning an ~-lmi~hlre of the whey material~ a charged polymer and a fatty acid.
Preferably the charged polymer is a cationic polymer. The charged polymer is added at a
concentration in the s~ llix~", c; wherein upon imposition of precipitation conditions the charged
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-3-
polymer forms a lipid-polymer precipitate and a liquid phase. The fatty acid preferably is
represented by the formula:
CH3 - (CH2)n - COOH
where n is a whole number from 4-10. The fatty acid is present in the ~.l,,,;x~ at a
concentration wherein upon imposition of precipitation conditions the fatty acid forrns a protein
precipitate and a liquid phase. The method further comprises the step of imposing l~lc;cil~i~Lion
conditions to form a protein precipitate, a lipid precipitate and a liquid phase. The liquid phase is
separated from the protein and lipid-polymer precipitates. This liquid phase is rich in
immlmnglobulins and can be further processed.
o Surprisingly and unexpectedly, the combination of a cationic polymer and a fatty acid
allows simlllt~neous precipitation of non-immllnoglobulin proteins and lipids to a degree that is
greater than when the cationic polymer and fatty acid are used sequentially. The rem~ining
eluant is >60% immunoglobulin. Such a purity is comparable with the concentration of
immllnoglobulins present in colostral whey and is comparable with the concentration and purity
necessary for achieving a conveniently sized dose forrn.
Also surprisingly and unexpectedly, as described in greater detail below immlmotherapy
products can be prepared which, in part as a result of the methods of the invention, can be
packaged in a unit dosage form of 1 gram or less and still contain effective amounts of antibody
for prophylaxis or trc~tment of active infection by pathogens in human subjects.The lipid precipitates and protein precipitates can be separated ~imlllt~neously by a
relatively low speed ct;~ ;rug~Llion (6-12, 000 x g), to produce a clear supernatant having a high
concentration of imml-n~globulins. The supern:~t~nt can be further concentrated and diafiltered
to produce a composition which is greater than 70% immllnnglobulin protein and less than 0.1%
lipid by dry weight. This supern~t~nt can be dried.
Preferably, the fatty acid and cationic charged polymer are selected to have precipitation
conditions which are similar.
Preferably, the cationic charged polymer is a selected from the group comprisingpolypeptides and charged polysaccharides. A preferred charged polysaccharide is chitosan.
c Chitosan is a cationic polymer derived from partially deacetylated chitin. Chitosan forms a gel-
like complex with polar lipids at a pH of 4.5 - 5Ø
Preferably, in the formula:
CH3 - (CH2)n - COOH
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. --4--
n is 6; that is, the fatty acid preferably is caprylic acid. Caprylic acid forms colloid-like
aggregates with non-imml-noglobulin proteins at a pH of 4.5-5Ø
In the situation where the cationic polysaccharide comprises chitosan, conditions for
forming a lipid precipitate can comprise a pH of 4.5- 5.0, a temperature of 20 - 25 ~C and a
concentration of chitosan of 0.05 to 0.3% by weight volume. In the situation where the fatty acid
is caprylic acid, conditions for forming a protein precipitate can comprise a pH of 4.5 - 5.0, a
telny~ldl~lre of 20-25 ~C and a concentration of caprylic acid of 1.0 to 5.0% by volume. The
coprecipitation of lipids and protein requires only one step and requires fewer reagents than
s~dLe steps. Indeed, surprisingly and unexpectedly, the chitosan-lipid precipitate aids in the
removal of the fatty acid-protein precipitate which by itself requires either lengthy high speed
(>l5,000 x g) cenkifugation or microfilkation for effective removal of the submicron size
particulates. Only "low speed moderate time "or "high speed short time" centrifugation is
n~ce,SS~ry for the removal of the combined precipitates. This capability significantly facilitates
large-scale m~m-f~turing. In the event additional purity is desired, the coprecipitation of lipids
and non Ig proteins with chitosan and caprylic acid can be repeated.
Preferably, the Ig rich sup~:rn~nt iS concenkated by ulkafiltration to remove low
molecular weight protein and peptides, forming a further Ig enriched retentate. Preferably,
ulkafiltration is performed with a membrane having molecular weight cutoff of about 10,000 to
150,000 Daltons, and, more preferably 20,000 to 50,000 Daltons.
Preferably, the Ig rich retentate is further concentrated by diafiltration to remove peptides,
minerals and lactose, to form a dialyzed immunoglobulin concentrate. A plert;lled dialysis
filkation buffer is a potassium cikate buffer of pH 6.5.
The immunoglobulin co~ -i--g supernatant is preferably processed by sterile filkation.
Sterile filkation is difficult with materials which have high lipid concentrations. The sterile
2s filkate is dried to form a dried immunoglobulin rich product.
Preferably, the dialyzed Ig concentrate is freeze dried to form a powder. The dried
immunoglobulin product has an improved shelf life since high lipid levels are a major factor in
dry product spoilage. The dried immunoglobulin products produced by the present method have
less than 6.0% lipid.
In certain embodiments, the methods can further comprise the steps of vaccinating a milk
bearing m~mm~l with one or more antigens that induce the production of antibodies against said
one or more antigen, then collecting milk or colostrum from said m~mm~l, and then processing
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the milk or colostrum to form the whey material. In one important embodiment, the antigen is
characteristic of enterotoxic Escherichia coli. According to a further aspect of the invention, the
antigen is one or more colonization factor antigens.
Embo-liment~ of the present invention are capable of using whey derived from
5 pasteurized cheese whey. Cheese whey is pasteurized at 161 - 163 ~F for 15 to 17 seconds, or
pasteurized for 30 minllte~ at 140~F - 142DF. In the alternative, the whey conc~llLldL/;: can be
pasteurized.
Preferably, the concentrated whey of the first adllli~ ; is made by ultrafiltering
pa~L~ d whey. Preferably, ultrafiltration is performed with a membrane having a 10,000-
o 150,000 Dalton molecular weight cut-off and, most preferably, a 30,000 Dalton molecular
weight cut-off.
Concentrated whey ofthe first ~hllixlllle may be prepared for example, by either spiral
membrane or hollow fiber ultrafiltration of pasteurized whey. Preferably, hollow fiber
ultrafiltration is used with membranes having a molecular weight cut off of 10,000-150,000
1S Daltons and, most preferably, 30,000 Daltons. A preferred hollow fiber ultrafiltration membrane
is a polysulfone hollow fiber membrane. This membrane produces a concenkation factor of 5-10
fold.
Preferably, the concentrated whey is further subjected to ion exchange chromatography to
reduce the concentration of non-imml-noglobulin proteins. A preferred chomotographic ion
20 e~ch~n~e process uses a strong anionic resin and whey protein concentrate having a pH of 6.5-
7Ø Anion exchange chromatography under these conditions can be used to remove from 20-
70% of non-immllnl-globulin proteins from whey or whey protein concentrate without
significantly altering immlmoglobulin levels. Partially deproteinized whey or whey protein
concentrates are a preferred starting material for whey immunoglobulin purification by the
25 combined precipitation process described above. Because of reduced non-immlln~globulin
protein levels and smaller processing volumes, reduced amounts of complexing agents are
therefore required with this staIting m~teri~l
Preferably, the antigen directed to ETEC comprises an antigen selected from the group
con~ tinp; of Colonization Factor Antigens (CFA). A plc~f~lled group of CFAs comprise
30 antigens of the CFA/I, CFA/II (CS 1, CS2, CS3) and/or CFA/IV (CS4, CS5, CS6) families. The
three most clinically prevalent families of CFA are identified in Table 1 below:
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TABLE 1
Colonization Factor An~igens of ETEC (CFAs)
Clinically Prevalent Famil;es of CFAs
FAMILY ANTIGENSIN FAMILY
(1) CFA/I CFA/I
(2) CFA/II CSl
CS2
CS3 (present in all CFA/II strains)
(3) CFA/IV CS4
CSS
CS6 (present in all CFA/IV strains)
Other CFAs of lesser clinical importance are: CFA/III (one member), CS17, PCF0159,
and PCF0166.
The antigen can be any one or more of the foregoing antigens. The antigen can consist,
for example, of at least CFA/I and CFA/II antigens or, more particularly, representatives of all of
the following antigens: CFA/I,CFA/II, and CFA-IV. A preferred CFA-II antigen consists of a
CS3 antigen. A ~lc;r~ d CFA-IV antigen is a CS6 antigen.
A further embodiment of the present invention features an immunoglobulin product20 derived from milk bearing m~mm~l~ hyperimmunized with an antigen to produce
irnmunoglobulins of interest. In one aspect of the invention the m~mmzll is hyperimmllni7~d
with an antigen directed to an ETEC/CFA to produce immunoglobulins for the treatment of
enterotoxigenic E. coli infections. The immunoglobulin product comprises at least 60% by
weight volume antibody, < 5% lipid, and < 20% non Ig proteins. This product can be further
~5 processed to remove water to produce an Ig product comprising at least 70% antibody, less than
or equal to 6.0% lipid and less than 20% non-Ig protein which can be ~lmini~iered for the
treatment of disease.
The irnmunoglobulin product also can comprise antibodies capable of binding antigens
f~om any number of sources including antigens characteristic of other pathogenic org~nism~.
30 Such pathogenic organism is preferably selected from one or more of the group consisting of
Cryptosporidium parvum, Rotavirus, Shigellaflexneri, Heliobacter pylori, Clostridium dif~icile,
Vibrio cholerae, Streptococcus mutans and Candida species Bacteriodes gingivalis, Bacteriodes
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melaninogenicus, Capnocytophaga species, Actinobacillus actinomycetemcomitans,
Porphyromonas gingivalis, Streptococcus so~rinus and enterotoxigenic Escherichia coli.
According to another aspect of the invention, the immlln~globulin product is isolated
from milk or colostrum and comprises antibodies capable of binding antigens associated with
ETEC. The antibodies exhibit a range of 3-50 fold higher titer than titers achieved by
inoculating bovines with a whole cell extract of enterotoxigenic Escherichia coli. These high
titers can be achieved by hy~ llulli~illg bovines with a vaccine comprising isolated, CFA
antigens preferably the majority of the antibodies in the immlm~globulin product that bind to
Escherichia coli bind to colonization factor antigens of Escherichia coli.
o According to another aspect of the invention, a passive immunotherapy product is
provided. It compri~es immunoglobulins that bind to enterotoxigenic Escherichia coli, packaged
in unit dosage form of 1 gram or less, and present in an arnount effective for treating active
infection in a human subject by or for prophylaxis of infection in a human subject by
enterotoxigenic Escherichia coli. Preferably the irnmunoglobulins bind colonization factor
s antigens as described above. In one embodiment the antigens are 'purified". The term
"purified", in the context of the present application, means substantially free of non-CFA
antigens. That is, such non-CFA antigens, if present, are not sufficient to create an immllne
response more than two-fold over baseline, or unimmunized state. Preferred CFA antigens are
CFA-I, CFA-II and CFA-III.
The immunoglobulin product of the present invention can be ~(1minist~red to subjects as
a reconstituted liquid, tablet, capsule, granules or food bar. Due to the removal of non-
imm~lnoglobulin proteins and lipids, an effective dose of immllnoglobulin can be ~-lmini.~tered
readily in a variety of formats. In one embodiment the effective dose is ~imini.ctered to an
individual infected with enterotoxic Escherichia coli or at risk of being infected with the same,
25 wherein the effective dose comprises antibodies that bind antigens of enterotoxigenic
Escherichia coli, the antibodies formulated as a product cont~ining at least 70%immlln--globulins, less than 6% lipid and less than 20% non Ig protein.
Embo~1iment~ of the present invention are capable of simultaneously removing theresidual lipids, cheese culture bacteria, denatured protein aggregates and fatty acid precipitated
30 whey protein. Lipid and protein precipitates can be removed by relatively low speed
cen~ ugation in the presence of the chitosan. The simultaneous centrifugation of the protein
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precipitates with the lipid precipil~L~s improved the recovery of highly purified
immlmoglobulins.
Protein and lipid precipitates are not readily removed by conventional microfilkation
methods without also reducing the recovery of immllnt)globulins because of the submicron size
of much of the fatty acid protein ~lecipi~l~. Although the concenkation of lipid in separated
whey is low, as whey is conc~;l,l,dled for procç~in~ residual lipids reach levels of 5-20% dry
weight.
These high lipid concenkations compLoll,ise the processing as well as the storage of
liquid Ig products. Products with high levels of lipids cannot be sterile filtered and are subject to
0 spoilage. These problems are overcome with the present process. Products made in accordance
with the present process feature low lipid and low non-Ig protein content. Such immunoglobulin
enriched products occupy a small volume and weight for an effective dosage. Ig fractions which
comprise the product can be sterile filtered and have a longer shelf life.
These and other features will become ~ from the drawings and the detailed
description which follows, which, by way of example, without limitation, describe pl~r~lled
embodiments of the present invention.
Brief Der~ ;~,lion of the Dl~wi~,
Fig. 1 depicts a flow diagram illuskating a method embodying features of the present
invention.
Figs. 2 and 3 graphically depict results from a clinical study in which subjects received a
product embodying features of the present invention.
Detailed Description of the Invention
The present invention is a method for isolating immunoglobulins from whey, including,
but not limited to, those directed to ETEC. Figure 1 which illuskates the method in a flow
diagram. Equipment for performing each step is well-known in the art.
As used herein, the term "whey" refers to the watery part of milk that separates from the
curds, as in the process of making cheese. "Whey fractions'- refers to a part of the whey
compri~inp: all or some of the whey proteins. "Partially deproteinized whey concentrate" refers
to a part of the whey in which all or some of the nonillll~ oglobulin proteins are removed. The
present method can be used to make an immunoglobulin product capable of being ~1mini~tered
in a relatively small dosage form.
-
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. _ g _
The process illustrated in Figure 1 begins with cheese mslking, and the products of whey
separation and clarification. The concentrated whey preferably is produced from a Swiss,
cheddar, mozzarella or provolone cheese making process. The whey originates from milk
produced by bovines, and preferably bovines which have been vaccinated with one or more
antigens. In one important aspect of the invention, the ~nim~l~ are immunized with CFAs.
Preferred CFAs are selected from CFA-I, CFA-II, and CFA-IV. Preferably, representatives of all
three CFA families are present in the vaccine. A preferred CFA-II is CS 1 and CS3 and, most
preferably, CS3. A I~er~ d CFA-IV is CS6. Animals immunized with such CFAs will secrete
within their milk immllnt~globulins that are directed to such antigens. Whey from one or more
~nim~l.c im mllni7t~.t1 with dirr~,lel~t antigens can be pooled to obtain imml-noglobulins that are
directed to a plurality of antigens. Typically, one would immunize all 7Jnim~l~ with all antigens.
Preferably, fat is separated from unconcentrated whey, obtained from cheese-making
processes, using a standard dairy crearn separator.
Preferably, the whey is concentrated by a factor of 5 to 1 0-fold over whey recovered
directly from cheese making processes. Preferably, the whey is concentrated by hollow fiber or
spiral membrane ultrafiltration depicted generally by the numeral 15 in Figure 1. A preferred
ultrafiltration process has a molecular weight cutoff of about 30,000 Daltons.
Preferably, the whey concentrate is pasteurized. Typically pasteurization conditions
comprise a Lelll~c;ldLule of 161-163 ~F for a period of time of 15-17 seconds or 140 - 142 ~F for a
period of time of 30 mimltes The whey can be pasteurized before or after concentration, using
similar pasteurization conditions.
A plefcll~d ultrafiltration process utilizes polysulphone hollow fiber membranes. During
the ultrafiltration process, a feed-to-permeation ratio of 5 to 1 is typical with a lumen feed
pressure of 25 to 40 psi, and an operating ~ e of 10-12~C.
Concentrated whey produced by ultrafilkation may be subjected to an optional ion~rch~nge chromatography step, illustrated in Figure 1 as pathway A. Preferably, the optional
chromatography step comprises the removal of an amount of non-immunoglobulin proteins with
an anionic exchange resin. This step is ~lesign~ted generally with the numeral 17. Following the
anion exchange process, the flow-through comprises an irnmunoglobulin enriched fraction.
Preferably, the immunoglobulins rich fraction is subjected to further ultrafiltration and further
ion ~ch~nge chromatography to remove additional non-immunoglobulin proteins. The
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- - 10 -
ulkafiltration step is de~ l generally with the numeral 19. These steps of ion exchange
chromatography and ultrafiltration can be repeated as desired.
Optionally, if fat is not removed by use of a cream separator prior to concentration of the
whey, then the Ig fraction is centrifuged for fat removal after final ion exchange chromatography
s and ultrafiltration. The step of centrifugation can be performed on the concentrated whey
product from an initial ultrafiltration step 15 as represented by ,~a~lw~y B. Preferably, a dairy
separator is utilized at 3,000 to 12,000rpm (5-15,000 x g) and the centrifugation is performed at a
temperature of 10 to 55~C. This step is generally designated by the numeral 23 in Figure 1. The
separated or delipidated whey constitutes a source of concentrated whey for the precipitation
treatments which follow.
A cationic polymer is selected to cooperate with an intencled fatty acid to undergo
precipitation of lipids as the fatty acid reacts with proteins. Cationic polymers are preferably
selected from the group comprising polypeptides or polysaccharides. A preferred polysaccharide
is chitosan. A particularly preferred type of chitosan is Seacure 443 (Pronova Biopolymers, Inc.,
1S Portsmouth, NH), a partially deacetylated poly-N-acetylglucosamine derived from shrimp.
An amount of chitosan effective to form a precipitate of residual lipids upon imposition
of precipitation conditions is approximately 0.2% by volume. The pH of this mixture is adjusted
to between pH 4.5 and 5.0 by the addition of a NaOH solution. This pH adjusted mixture is then
further reacted by the addition of a fatty acid, preferably caprylic acid, which reacts with proteins
as the chitosan polymer reacts with lipids.
A preferred polypeptide is selected from the group consisting of basic polyamino acids or
acid soluble basic proteins such as type A Gelatin (pI 7.0-9.0). These polypeptides are capable
of forming a lipid precipitate at a pH between 4.5 and 5.0 at concentrations of 1-5% by weight
polypeptide.
An effective amount of caprylic acid, to form a protein precipitate upon imposition of
precipitation conditions is approximately 5% by weight volume. Mixing of chitosan, caprylic
acid and concentrated whey may comprise the use of stirrers, paddles or other mixing apparatus
known in the art. Lipid precipitation conditions for chitosans and lipids, comprise a temperature
of 20 to 25 ~C and a pH of 4.5 to 5Ø Protein precipitation conditions, for non-IgG proteins and
caprylic acid, comprise a temperature of 20 to 25~C and a pH of 4.5 to 5Ø Thus, lipid
plecipit~tion conditions and protein precipitation conditions may be imposed simultaneously.
-
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'- - 11 -
Preferably, lipid precipitdtion conditions and protein precipitation conditions are imposed
for 5 to 30 minutes after the ~(l.,,ix~ is formed by mixing. That is, a period of 5 to 30 minutes
is allowed for the ~ e to stand substantially motionless, with a 15 minute period preferred.
An ~ro~liate centrifuge capable of receiving the ~fimixtllre co~ i "i t-~ a lipid
5 precipitate and a protein precipitate is used to separate the solid and liquid phases. The
centrifuge should be capable of subjecting the mixture to a force of 15-20,000 x g and preferably
an ejecting solid centrifuge. A ~ led bowl centrifuge is a Sharples centrifuge and a preferred
ejecting centrifuge is an automatically desludging Carr P-12 centrifuge or Alfa-Laval centrifuge.
Preferably, the centrifuge assembly is m~int~ined at a telllpeldlu.e of 20 to 25 ~. Under these
o conditions the centrifuge se~dles the lipid precipitate and protein precipitate from the
immunoglobulin rich supern~t~nt The lipid precipitate, comprising lipid and chitosan, aids in
the removal of the protein precipitate allowing low gravity forces to remove substantially all of
the colloidal particles. This ~leeipikllion step also substantially reduces bacterial co~ ion~
rrims~rily due to flocculation of c~ ",;"~tin~ microbes by chitosan. Following centrifugation
15 the pH of the liquid supern~t~nt fraction is adjusted to 6.5. The coprecipitation of lipids and non-
IgG proteins by chitosan and caprylic acid and centrifugation may be repeated if desired.
The irnmunoglobulin rich supern~t~nt can be subjected to further concentration. This
concentration is performed by ultrafiltration represented generally by the numeral 31 in Figure 1.
Preferably, ultrafiltration is performed using polysulphone hollow fiber membranes having a
20 molecular weight cutoffof 10,000-150,000 Daltons and most preferably 30,000 Daltons. This
ultrafiltration step can produce a fu~ther concentration of the supernatant by 15 to 25-fold. The
ultrafiltration allows further permeation of low molecular weight polypeptides through the
membranes while r~L~ g an immlmoglobulin rich retentate. Typically, the ultrafiltration has a
feed-to-permeation ratio of 5:1, a lumen feed pressure of 15 to 30 psi, and is m~int~ined at a
25 temperature of 10-12 ~C.
The immllnoglobulin rich retent~tP can be further diafiltered using a polysulphone hollow
fiber membrane having a nominal molecular weight cutoff of 10,000- 150,000 Daltons or most
preferably, 30,000 Daltons. The retentate is diafiltered ~ltili7ing a 15 mM potassium citrate
buffer in demineralized tap water at a pH of 6.5. The diafiltration allows further permeation of
30 polypeptides, minerals and lactose. Diafiltration is performed with a permeation ratio of 5: 1, a
lumen feed pressure of 15 to 30psi, and a temperature of 10-12~C.
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- -12-
A final ret~nt~t~ from the diafiltration is dried by either freeze drying or spray drying.
This step is generally ~leci~n~tecl by the numeral 33 in Figure 1. The dry powder is characterized
as at least 85% protein, which protein represents 70% pure Ig and less than 6% lipid by weight.
A ~l~r~llc;d method of ~lmini~tration comprises enteric coated tablets, capsules, or
s pellets. Individuals skilled in the art are able to form~ t~ the IgG product into one or more
enteric coated tablets, capsules, and pellets. There are many possible enteric formulations. One
formulation is set forth below:
Enteric-Release IgG granules
Ingredient In eachIn 10,000
0 Milk Protein 372 mg 3,720 g
Avicel PH101 79.9 mg 799 g
Ac-Di-Sol 26.7 mg 267 g
Polyglycol E 4500NF 53.4 mg 534 g
HPMC 14 mg 140 g
EudragitTM L30D 140 mg 1400 g
Triethyl citrate 14 mg 140 g
Weight of granulation 700 mg 7,000 g
In the above formulation, the first three ingredients are blended to uniformity. The fourth
ingredient is dissolved in water, and added to the uniforrn blend of the first three ingredients to
form a wet granulation. The wet granulation is extruded and spheronized into 1.0-2.0 mm
granules. The granules are dried to ~ o~illldlely 2% moisture. A dispersion of hydroxypropyl
methyl cellulose (HPMC) in water is applied to the granules in a fluidized bed spray dryer. A
dispersion of the EudragitTM L30D (Rohm Pharma, Basel, Switzerland) and triethyl citrate NF
(Morflex Inc., Greensboro, NC) in water is applied to the HPMC coated granules in a fluidized
bed dryer.
In the above formulation, the milk protein would comprise a purified IgG product. In the
above formulation, Avicel PH101 (FMC Corp., Newark, DE) is a binder. Avicel PH101 is a
microcrystalline cellulose. Other binders may be substituted for microcrystalline cellulose.
In the above formulation, Ac-Di-Sol (FMC Corp., Newark, DE) is a disintegrant. Ac-Di-
Sol is a cross-linked sodium carboxymethylcellulose. Other disintegrants may be substituted for
Ac-Di-Sol in the above formulation.
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In the above formulation, polyglycol E4500 NF (Dow Chemical, Midland, MI) is a
diluent. Polyglycol E4500 is a polyethylene glycol of average molecular weight of 4500. Other
diluents may be substituted for polyglycol E4500.
In the above formulation hydro2sy~l0~yl methyl cellulose (HPMC) (Colorcon, West
5 Point, PA) is a film coat. EudragitTM L30D is an enteric polymer. EudragitTM polymers
comprise copolymers of methacrylic acid and ethyl acrylate or methacrylic acid and methyl
methacrylate. Triethyl citrate is a plasticizer. Other enteric coatings, polymers and plasticizers
may also be used. Preferably, the coatings, polymers and plasticizers allow the granules to
survive gastric acid for two hours. Preferably, the coatings, polymers and plasticizers allow
10 dissolution and release of the immnnoglobulin product at a pH of 5 or above.
These and other features will be apparent from the following examples which further
hi~hlight important aspects of the present invention.
F~ ples
e 1 - Production of ETEC ant~ens ~-~d preparation of ~nti-~TFC
immnno~lobu~
This Example features the making of an IgG product with activity against enterotoxigenic
E. coli (ETEC).
Materials a~d Methods
Bacterial strains for vaccine production. Enterotoxigenic E. coli strains used in the
20 manufacture of this product were obtained from the culture collection of the Center for Vaccine
Development at the University of Maryland at Baltimore, the Walter Reed Army Institute for
Research or University of Texas at Houston. Strains M424C 1 (CS 1, CS3) and E9034A (CS3)
(UM-Baltimore) were used for production of CS1 and CS3; strain H10407 (UM-Baltimore) was
used for production of CFA-I. Strain M295 (W.R.A.I.R.), or CID553 (UT-Houston) is used for
2s the production of CFA-IV.
Production of Bovine Vaccines
F,xpression of CFA/I. CS3~ and CS6 from native or~ni~ in broth cultures. Stock cultures
..
were m~int~ined in the frozen state at -80 ~C. Frozen cells were used to inoculate plates of CFA
agar (lOg/L Ç~c~mino acids, 6 g/L yeast extract, 50 mg/L magnesium sulfate, 5mg/L m~n~ne~e
30 chloride, 15 g/L agar) which were incubated overnight at 37~C. On the following day, cells were
aseptically scraped from the plates and resuspended in phosphate buffered saline, pH 7.2. This
cell suspension was used to inoculate sterile CFA broth (10 g/L ç~nnin~ acids, 6 g/L yeast
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-14-
extract, 50 mg/L mslgn~sium sulfate, 5 mg/L m~ng7n~se chloride) prewarmed to 37~C for
fermentation. Preferably, sufficient quantities of the cell suspension are added to bring the
optical density of the starting broth to 0.05-0.08 at 660 nm ~O.D.660). After inoculation, the
culture was aerated, preferably by mixing at 50-70 rpm under 20-30 psi positive air pressure in a
5 stainless steel, waterjacketed ferment~r. Cell growth was monitored continuously by
spectrophotometry, and the cells were harvested just prior to early stationary phase growth,
preferably at an O.D.660 = 0.8 - 1.2, and preferably after 4-5 hours of growth.
Cells were harvested and concentrated 40-50 fold, preferably using tangential flow
filtration with a 0.1 ,um pore sized, low protein-binding membrane.
lo P ~rification of CFA/L CS3, and CS6 from native or~-ni~ms ;n broth cultures. CFAs were
sheared from the surface of the concentrated cells, preferably using continuous flow sonication at
4~C for 30-45 minutes/L concentrate using a flow rate of 150-200 ml/min.
Cell debris was removed by centrifugation, preferably at 10,000-15,000 x g for 20-30
mimlt(~
Ammonium sulfate was added to the CFA-rich supernatant to 10-20% saturation and
incubated at 4~C with stirring for at least 30 minlltçs followed by centrifugation preferably at
15,000-20,000 x g for 20-30 minutes to remove non-CFA proteins. Additional ammonium
sulfate was then added to the sup~ to 40-50% saturation and stirred at 4~C for at least 60
minlltes followed by centrifugation preferably at 15,0.00-20,000 x g for 20-30 minutes to collect
20 CFA proteins. The CFA-rich pellet was resuspended in 50 mM phosphate buffer, pH 7.5 (PB).
Ammonium sulfate was removed from the CFA suspension by dialysis, preferably
against 5,000-10,000 volumes of PB using 10,000-14,000 MW Spectrapor (Spectrum Medical
Industries, Inc., Houston, TX) tubing at 4~C.
The dialysate was purified by either ion exchange chromatography (CFA/I) or size25 exclusion chromatography (CS3 or native CS6). Taking advantage of the large size of the CFA
polymers, relatively pure CFAs elute in the void fraction in each case.
For CFAII, radial flow chromatography is the method of choice using dimeth~vl amino
ethyl substituted (DEAE) cellulose, preferably cross-linked for support. After equilibrating the
column with PB by standard methods, the dialysate was run over the column, preferably 1 mg of
30 protein per 2-6 ml of resin is applied at a flow rate of 50-70 ml/min. Elution of the CFA-rich
void fraction was observed by measuring the absorbance of the column effluent at 280 nm by
standard methods.
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For CS3 and native CS6, size-exclusion chromatography is the method of choice using
acrylamide cross-linked dextran beads, preferably with a molecular size fractionation range of
10,000-1,500,000 using an axial column. The column was equilibrated with PB cont~inin~ a
chaotropic agent, preferably N-lauryl sarcosine (10-40 mM), by standard methods. The resulting
s dialysate was then run over the column, preferably such that 1 mg of protein per 8-12 ml of resin
is applied at a flow rate of 8-12 ml/min. Elution of the CFA-rich void fraction was observed by
measuring the absorbance of the column effluent at 280 nm by standard methods.
~ression of CS6 from recombin~nt or~ni~m~ in broth cultures. Stock cultures were" ,;~ P-l in the frozen state at -80 ~C. Frozen cells were used to inoculate plates of Luria Broth
0 (LB) agar (10 g/L tryptone, S g/L yeast extract, S g/L sodium chloride, 15 g/L agar) which were
incubated overnight at 24-28 ~C. Preferably, the genes encoding the recombinant CFA are found
on an extrachromosomal element (plasmid) which also expresses proteins co~lfe";~g resistance
to a particular antibiotic. To ensure the stability and increase the copy number of the plasmid,
25-50 ~Lg/ml of the a~,~p,iate antibiotic were included in all growth media. (For strain M295,
the antibiotic is ampicillin). On the following day, cells were aseptically scraped from the plates
and resuspended in phosphate buffered saline, pH 7.2. This cell suspension is used to inoculate
sterile LB broth (10 g/L tryptone, S g/L yeast extract, 5 g/L sodium chloride) cont~inin~ 25
,~Lg/ml ampicillin and prewarmed to 24-28~C for fermentation. Preferably, sufficient quantities
of the cell suspension are added to bring the optical density of the starting broth to 0.06-0.10 at
660 nm (O.D.660). After inoculation, the culture was aerated, preferably by mixing at 40-60 rpm
under 5-10 psi positive air pl~s~,ule in a stainless steel. waterjacketed fermenter. Cell growth
was monitored continuously by spectrophotometry, and the cells were harvested just prior to
early stationary phase growth, preferably at an O.D.660 = 0.8 - 1.4, and preferably after 4.5 hours
of growth.
Cells were harvested and removed as the majority of the recombinant CS6 antigen is shed
into the broth. The cells are removed, preferably, using tangential flow filtration with a 0.1 llm
pore sized, low protein-binding membrane. The supernatant was then concentrated 100-200 fold,
preferably using a hollow fiber filtration system using a polysulfone membrane with a molecular
weight cutoff of 30,000 Daltons. The concentrate was then diafiltered against PB to remove
media components. Typically, the diafiltrate itself is of suff1cient purity that no further
purification is necessary. If purification is necessary, the size exclusion chromatography scheme
outlined for CS3 above is llti~
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Pl~dLions in which greater than 70% of all Coomassie-Brilliant Blue staining protein
was CFA were considered acceptable for use as bovine vaccines. The CFAs were concentrated
by diafiltration on a stirred cell under nitrogen gas and sterilized by passage through a 0.45
micron syringe filter. All vaccine ~lc;~dLions were tested for bacterial and fungal sterility. and
5 the presence of <100,000 EU/ml of endotoxin as lletermined by limulus lysate assay (Bio
Whittaker). The final vaccine was prepared by mixing the ~p~ ,liate dose of antigen 1:1 (v/v)
with a synthetic non-LPS co~ ;llillg adjuvant. A preferred adjuvant is a Freund's adjuvant or its
synthetic equivalent.
l~ovine v~çi~tions. All vaccinations were perforrned under USDA approval and ~tlmini~tered
10 under the direction of a licensed veterinarian. All ~nim~l~ used were healthy Holstein dairy
cows. Health records were m~int~ined and only healthy, mastitis-free ~nimz~l~ were included in
the study. A series of three intramuscular vaccinations were a-lministered deep into the rear
thigh muscle. A total volume of two ml was ~-lmini~tered at a single site and the ~nim~l~ were
monitored for adverse reaction. None were observed. Vaccinations were given three weeks
1S apart, and milk collected regularly beginning one week after the third shot. Milk samples were
taken from every batch and shipped frozen to ImmuCell corporation (Portland, ME) for ELISA
analysis. Although the anti-CFA titers for each batch were known, no attempt was made to use
only the highest titer milk for production of anti-enterotoxigenic E. coli imrnunoglobulin
(AEMI). To cim~ te continuous production, milk from seven different batches, collected over a
20 four week period, were pooled to make the clinical test material described herein.
Vaccinations were performed separately with CS1, CS3 and CS6, alone and in
combination with equally successful results.
Preparation of anti-E. coli Bovine Milk Immuno~lobulin. Hypt;~ lulle milk was processed
into provolone or mozzarella cheese by standard dairy practices. The aqueous whey fraction
25 collt~ immunoglobulins was clarified and separated using standard dairy whey
centrifugation methods. Clarified whey was first pasteurized by heating of 1 59~F for 15 sec.
using a standard dairy HTST pasteurizer. The heat treated whey was concentrated sixfold (6X)
using hollow fiber membranes with a molecular weight cut off of 30,000 Daltons. Concentrated
whey was enriched in immunoglobulins by anion exchange chromatography using an ISEP
30 Chromatography System (Advanced Separation Technologies, Lakeland, Florida) in a process
generally depicted as pathway A. See Figure 1. Whey concentrate in this procedure is first
adjusted to pH 6.8 by addition of a NaOH solution and passed over 1 0xl 00 cm columns
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cont~inin~ a qn~tern~ry ammonium substituted poly~Lylel1e resin. Resin was first washed and
pre-equilibrated to pH 7.0 with dilute buffer. Non-immllnnglobulin proteins were absorbed
under these conditions while the flow-through fraction was enriched in immlm~globulins.
The flow-through fraction was then concentrated by hollow fiber filtration (A/G
5 Technology Corp., Nee~lh~rn, MA), using polysulfone filtration cartridges (30,000 MW cut off,
24m2 surface area). The rçsllltin~ flow-through concentrate was centrifuged to remove excess
non-polar lipids.
~ em~ininp phospholipids and residual non-Ig proteins were then precipitated by
sequential addition of the flocculating agents chitosan (Sea Cure 443, Pronova Biopolymers,
0 Inc., Portsmouth, NH) and caprylic acid (Henkel, Emersol 6357). The precipitation reaction was
carried-out using chromatographically deproteinized and defatted whey at a temperature of 20-
25 ~C. Chitosan was added to a final concentration of 0.2% and the pH of the mixture adjusted to
pH 4.6. Caprylic acid was added to a final concentration of 5% by volume and the mixture
stirred for 5 and followed by 15 to 30 minutP~ static incubation.
The resulting ~l~ci~ L~ was removed by centrifugation in a Sharples Centrifuge (Model
AS-16, Alfa-Laval, W~rmin.~t~r, PA) and the ~uy~ adjusted to pH6.5 by the addition of
NaOH. The centrifugation supernatant was concentrated to approximately 5% solids using a
hollow fiber filtration system. After concentration, residual lactose, milk peptides and other salts
were removed by step-wise diafiltration against three volumes of 15 mM potassium citrate pH
20 6.5.
The buffered immunoglobulin fraction was subsequently lyophilized to produce a f~mal
powder. Analysis of a reprçsent~tive lot of anti-E. coli immunoglobulin produced by this
procedure revealed that the lyophilized powder contained 78% protein, 5.5% fat, 1.1%
carbohydrate, 10.5% ash due to added potassium citrate buffer, 2.2% residual ash and 2.7%
2s moisture. Ig comprised 79% of the total protein as revealed by sç~nning densitometry and SDS-
polyacrylamide gel electrophoresis. ~dditional milk proteins present included beta-
lactoglobulin, alpha-lactalbumin, serum albumin, and trace amounts of casein. Table 1 below
describes the recovery of immlln~globulin activity and the Ig purity at different stages in this
purification process.
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T~BLE 1
Summary of Anti-E. coli Immunoglobulin Product Purification
Anti- %
E. coli Antibody %
Process Activity Volume Activity Ig/Total
Intermediate (U/ml) (L) Recovery Protein
Pasteurized Whey 28 3800 100% 1.57%
6X Whey Concentrate 160 633 95.3% 2.29%
Chromatographic
0 Flow Through 108 783 79.8% 8.85%
Defatted Concentrate 1861 45.6 79.9% 11%
Ig Concentrate 4750 11.4 51.1 % 79%
Qu~ntitation of anti-CFA activity in ETEC Directed Product by ELISA. Anti-CFA titers
15 were determined by measurement of binding of milk antibodies to purified antigen-coated plates
by ELISA using standard methods. The absolute ELISA titer or OD is variable and dependent
primarily on the antigen plepal~ion used to coat the wells. Thus, the most accurate and
me~ningful comparison of multiple samples was made by establishing a reference standard from
which all unknown samples titers were interpolated. Dilutions of our anti-CFA milk standard
20 were run on each plate cont~ining unknown samples and a standard curve was constructed.
Titers for unknown sarnples were then interpolated from the standard curve. This norm~li7P~l all
ELISA data and permitted me~nin~ful comparisons between samples run on different assays to
be made.
The ETEC product of the present invention derived from bovines hyperirnmunized with
25 purified CFAs exhibited titers which were 3-10 fold higher than titers derived from products
derived from bovines immllni~d with whole cell extracts. These results are set forth in Table 2
below:
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. - 19-
TABLE 2
2 3
VaccineFoldIncreaseRatio (Ag/WC)
Whole Cell12.2
2 CFA/l[ 82.8 6.8
s 3 CS3 42.4 3.5
4 CS6 46 3.8
5 CSl 61.1 5.1
0 Voll~nteer study. Twen~y-five healthy adult volunteers, housed as in-patients in the isolation
ward at the Center for Vaccine Development (University of Mar,vland School of Medicine), were
randomly assigned to three groups. Placebo (n=10), High dose (n=l 1), and Low dose (n=4) by
~ssi~ning subject ID numbers to identically packaged foil pouches co"l~ -g measured doses of
each test article. All investigators and volunteers were blinded to these tre~tment group
~ssignments throughout the study and during ~s~essment of outcome. Each placebo pouch
cont~ined a single dose (1.7g) of Lactofree(~ (Mead Johnson), a lactose-free infant formula. The
high dose pouch of product contained 1 .7g (lg IgG) and a low dose pouch product 0.43g (0.25g
IgG) respectively.
All 25 individuals received three doses/day of either the placebo or one of the two doses
zo of product after meals for two days. Each dose consisted of the assigned pouch of product as a
powder dissolved in 8 ounces (240 ml) of water cont~ining 2g of sodium bicarbonate. On day
three, two hours after consnmin~ the morning dose, all volunteers drank 4 ounces (120 ml) of
water cont~ininp 2g of sodium bicarbonate. One minute later, each received an oral challenge
inoculum ct)nt~ining 109 of Hl 0407 (078:H11), a CFA/I-bearing ETEC strain suspended in 1
25 ounce (30 ml) of water cont~ining sodium bicarbonate. Fifteen minutes later, a second dose of
either product or placebo was given followed by the two normal afternoon doses. On days 4-7,
three doses/day were ~lministered as before after each meal.
Volunteers collected every bowel movement produced during the study which were
graded for consistency, weighed and logged to record number produced per day by an attending
30 nurse. Daily stool samples were taken for bacteriology ex~min~tion.
Daily medical rounds were conducted to monitor development of any abnormal
symptomology. Before being discharged from the hospital, all volunteers were given a three day
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course of ciprofloaxcin (500 mg b.i.d.) to eradicate the challenge org~ni~m The primary
effectiveness variable was the clinical diagnosis of ~ rrh~ defined as one liquid stool of 300 ml
or more or two liquid stools totaling 200 ml during any 48-hour period within 120 hours after
challenge.
s Clir ical. Seven out of the 10 volunteers in the placebo group presented with diarrhea after
challenge compared to only one out of fifteen volunteers in the groups receiving the ETEC
Product . The results are depicted in graph form in Figure 2. Clinical Results From Phase I/II
Study, "Protection Against Oral Challenge of Enterotoxigenic Escherichia coli (ETEC) using
Prophylactic Hyp~ l"~ e Tmmllnoglobulin in Healthy Normal Volunteers". The primary
effectiveness variable defined for the study described above was the clinical diagnosis of ~ rrhe~
defined as one liquid stool of 300 ml or more within a 120 hours of challenge with ETEC, or at
least two liquid stools of 200 ml or more. Total patients are indicated with bars with bold dots
widely spaced. Patients receiving a placebo high dose and low dose are ~ n~d by bars with
small dots with fine spacing. Each bar is separately labeled for placebo, high does and low dose.
1S Co, - .p~. h~ the attack rate in the placebo group (70%) with the attack rate in the treated groups
(6.7%), prophylactic ~lmini~tration of ETEC product brought about a 90% protection rate. The
mean stool volume in volunteers with ~ rrhe~ was 1327 ml (range = 263-4421), and the mean
number of stools was 7.4 (range = 2-21). The mean incubation time was 58.8 hours (range =
19.4-100.3 hrs.).
In addition to ~ rrhe~ daily medical rounds were conducted to record the incidence of
several other symptoms. Anorexia was reported by 6/10 controls compared to the 1/15 treated.
Malaise was found in 3/10 controls and 1/15 treated. Five out of 10 controls reported stomach
gurgling compared with 2/15 for the treated. Five out of 10 controls experienced headaches
compared to 4/15 for the treated. Finally, while all 10 volunteers receiving the placebo
experienced abdominal cramps, only 2/15 volunteers receiving the ETEC product did. No
adverse side effects were observed in any volunteer. These results are depicted in bar graph form
in Fig. 3. Figure 3 depicts the number of patients exhibiting various symptoms vs. the total
number of patients in two control groups. The first control group received a placebo. The
second control group received the ETEC Product. Total patients are depicted in bars with bold
dots with wide spacing. Patients exhibiting symptoms of anorexia are depicted with bars with
fine dots with wide spacing. Patients exhibiting C~ g symptoms are depicted with bars with
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-21-
light cross h~tçhin~Q;. Patients exhibiting symptoms of gurgling are depicted with bars with dark
cross h~t-~hinp .
Bacteri~lo~v. Daily stool samples were analyzed for the presence of the challenge organism to
e shedding over time. The average number of challenge orgs~ni~m~ per gram of stool
5 shed by volunteers receiving the placebo at the time of m~im~l shedding was 4.5 x 1 o8 CFU/g.
The peak mean value for volunteers receiving the ETEC product was 6.2 x 1 07/g. The average
number of days that volunteers excreted the challenge organism was virtually the same between
groups: 5.3 days for controls verses 5.4 days for volunteers receiving the ETEC product (range =
4-6 days).
Thus, the present methods provide for large scale production and purification of CFA and
the use of such CFAs to produce a milk-derived product effective in treating enterotoxigenic E.
coli disease. This milk-derived, immllnoglobulin concentrate has specific activity against
purified colonization factor antigens. The product was well tolerated and no adverse reactions
were reported. Antibodies against CFAs are sufficient for protection, and are an ~Itern~tive to
15 ç~ tinp drug interventions.
Example 2 - Prepara~ion of Anti-Gy~lo~oridium parvum Immunoglobulins
Hypel;.,...~l-..e milk from cows immllni7~cl with a killed C. parvum vaccine wasprocessed into provolone or mozzarella cheeses by standard cheese making procedures. The
aqueous whey fraction cu--l~ i--g immlln~globulins was clarified and separated using standard
20 whey centrifugation methods. The schematic for anti-C~yprosporidium immlmf)globulin
purification in this example is shown in Figure 1.
Using pa~Leu~ ion and hollow fiber ultrafiltration procedures described for initial
immllne whey processing in Example 1, a 6X whey concentrate was prepared and subjected to
direct chitosan/caprylic tre~tment as outlined in Figure 1 (pdlhw~y B). Chitosan was added to a
25 final concentration of 0.15% by weight while stirring the whey concentrate. The pH of this
mixture was adjusted to pH 4.9 by the addition of an NaOH solution after which caprylic acid
was added to a final concentration of 4.0% by volume with mixing. The chitosan and caprylic
precipitation reactions proceeded at 23~C for 30 minutes with int~nittent stirring.
The chitosan-lipid and caprylic-protein precipitates were separated by centrifugation in a
30 Sorvall centrifuge at 10,000 x g and the resulting sup~rn~t~nt adjusted to pH 6.5 by the addition
of NaOH. Analysis of anti-Cryptosporidium antibody activity was carried out using standard
sandwich ELISA procedures with C. parvum antigens coated on microtiter plates.
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Ig purity at different steps was determined by densitometric sc~nning of 4-20% SDS-
PAGE gels run under non-refl~ ing conditions at pH 8.5 which were stained with Coomassie
Blue. The results of this purification are shown in Table 2 below.
TABLE 2
s S~ ry of Anti-C)yptosporidiumparvumImmunoglobulin Purification
Anti- % %
Crypto Antibody Purity
Process Activity Activity Ig/Total
Tnt~rmerli~te (U/ml) Recovery Protein
Raw Whey 824 100 6.7
Pasteurized Whey 761 92.4 6.7
6X UF Whey
Concentrate 4611 100 8.2
Chitosan/Caprylic
Supern~t~nt 3476 75.5 66.7
20 li,Y~n~ple 3- Preparation of Rotavirus Immuno~.lobulins
This Example describes making an Ig product for preventing or treating Rotavirusinfections. Cows would be immllni~d with a vaccine cont~ining killed virus or purified viral
neutralization antigens (eg. G or P antigens) representing the four major rotavirus types (1-4)
infective for humans. H~cl;",."l-ne milk would be processed into provolone or mozzarella
25 cheese by standard dairy practices. The aqueous whey fraction cont~ining immllnnglobulins
would be clarified and separated using standard dairy whey centrifugation methods. Clarified
whey would be first pasteurized by heating of 1 61~F for 15 sec. using a standard dairy HTST
pasteurizer. The heat treated whey would be concentrated sixfold (6X) using hollow fiber
membranes with a molecular weight cut off of 30,000 Daltons. Concentrated whey would be
30 enriched in immunoglobulins by anion exchange chromatography using an ISEP
Chromatography System (Advanced Separation Technologies) in a process generally depicted as
p~lhw~y A of Figure 1. Whey concentrate in this procedure would be first adjusted to pH 6.8 by
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addition of aNaOH solution and passed over 10x100 cm columns C(JI~t~illillg a quaternary
ammoniurn substituted polystyrene resin. Resin would be first washed and pre-equilibrated to
pH 7.0 with dilute buffer. Non-immlmoglobulin proteins would be absorbed under these
conditions while the flow-through fraction would be enriched in immunoglobulins. In the
S ~ltt~rn~tive, a process depicted in pathway B of Figure 1, and described in Example 2 can be
tili7.~1
The flow-through fraction would be then concentrated by hollow fiber filtration (A/G
Technology), using polysulfone filtration c~c~ettes (30,000 MW cut off). The resulting flow-
through concentrate would be centrifuged to remove excess non-polar lipids.
Rem~inin$~ phospholipids and residual non-Ig proteins would be precipitated by
sequential addition of the flocculating agents chitosan (Pronova, Inc.) and caprylic acid. The
precipitation reaction would be carried-out using chromatographically deproteinized and defatted
whey at a temperature of 20-25 ~C. Chitosan would be added to a final concentration of 0.2%
and the pH of the llliX~ adjusted to pH 4.6. Caprylic acid would be added to a final
1S concentration of 5% by volume and the mixture stirred interrnittently for 30 minlltes
The resulting precipitate would be removed by centrifugation in a Sharples Centrifuge
(Alfa Laval, Model AS-16) and the supernatant adjusted to pH6.5 by the addition of NaOH. The
cc;~ irugation supernatant would be concentrated to approximately 20% solids using a hollow
fiber filtration system. After concentration, residual lactose, milk peptides and other salts would
20 be removed by step-wise diafiltration against three volumes of 15 mM potassium citrate pH 6.5.
The buffered immunoglobulin fraction would be subsequently lyophilized to produce a
fmal powder. Such antibodies purified from whey by the procedures described can be
incorporated into foods or drinks to prevent rotavirus infections in young children and older
adults.
25 F,~ rle 4 - Preparation of Shi~ella ~lexneri Immuno~lobulin
This Example describes making an Ig product for preventing or treating Shi~ella flexneri
infections. Cows are immlmi7~cl with a vaccine cont~ining killed bacteria or purified cell wall
antigens together with inactivated Shigella toxins. Hyperimmune milk would be processed into
provolone or mozzarella cheese by standard dairy practices. The a~ueous whey fraction
30 co~ i"i~g immunoglobulins would be clarified and separated using standard dairy whey
centrifugation methods. Clarified whey would be first pasteurized by heating of 161 ~F for 1
sec. using a standard dairy HTST pasteurizer. The heat keated whey would be concentrated
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-24-
sixfold (6X) using hollow fiber membranes with a molecular weight cut off of 30,000 Daltons.
Defatted whey would be enriched in immunoglobulins by anion exchange chromatography using
an ISEP Chromatography System (Advanced Separation Technologies) in a process generally
depicted as pathway A of Figure 1. Whey concentrate in this procedure would be first adjusted
to pH 6.8 by addition of a NaOH solution and passed over 1 0xl 00 cm columns co~ ;rlg a
qll~t~rn~ry ammonium substituted poly~iylclle resin. Resin would be first washed and pre-
equilibrated to pH 7.0 with dilute buffer. Non-immunoglobulin proteins would be absorbed
under these conditions while the flow-through fraction was enriched in immlln~globulins. In the
alternative, pathway B - a process depicted as described in Figure 1, and Example 2, can be
10 lltjli7.~.'1
The flow-through fraction would be then concentrated by hollow fiber filtration (A/G
Technology), using polysulfone filtration c~settes (30,000 MW cut off). The resulting flow-
through concentrate would be centrifuged to remove excess non-polar lipids.
R~m~ining phospholipids and residual non-Ig proteins would be precipitated by
15 sequential addition ofthe flocculating agents chitosan (Pronova, Inc.) and caprylic acid. The
~le~ iLation reaction would be carried-out using chromatographically deproteinized and defatted
whey at a temperature of 20-25 ~C. Chitosan would be added to a final concentration of 0.2%
and the pH of the mixture adjusted to pH 4.6. Caprvlic acid would be added to a final
concentration of 5% by volume and the mixture stirred intermittently for 30 minlltes
The resulting precipitate would be removed by centrifugation in a Sharples Centrifuge
(Alfa Laval, Model AS-16) and the sup~ t~nt adjusted to pH6.5 by the addition of NaOH. The
centrifugation supern~t~nt would be concentrated to approximately 20% solids using a hollow
fiber filtration system. After concentration, residual lactose, milk peptides and other salts would
be removed by step-wise diafiltration against three volurnes of 15 mM potassium citrate pH 6.5.
2s The buffered irnrnunoglobulin fraction would be subsequently Iyophilized to produce a
final powder. Antibodies to these antigens which are present in whey can be purified by the
procedures described and ~rimini.~tered in food, drink or capsule/tabled from for the prevention of
Shigella infections arnong susceptible or exposed individuals.
~Y~n~ple 5 - Preparation of Heliobacter pylori Immuno~loloulins
This Example describes making an Ig product for preventing or treating Heliobacter
pvlori infections. Cows would be imm1-ni7ed with purified antigens of H.pylori represented by
presumed virulence factors such as urease, vacuolating cytoxins and flagella which are thought to
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be important in bacterial infection of gastric mucosa. Hyp~ l""ul,e milk would be processed
into provolone or mozarella cheese by standard dairy practices. The aqueous whey fraction
co~ lillp; immllnoglobulins would be clarified and separated using standard dairy whey
centrifugation methods. Clarified whey would be first pasteurized by heating of 161~F for 15
sec. using a standard dairy HTST pasteurizer. The heat treated whey would be concentrated
sixfold (6X) using hollow fiber membranes with a molecular weight cut off of 30,000 Daltons.
Concentrated whey would be enriched in immlmQglobulins by anion exchange chromatography
using an ISEP Chromatography System (Advanced Separation Technologies) in a process
generally depicted as pathway A of Figure 1. Whey concentrate in this procedure would be first
10 adjusted to pH 6.8 by addition of a NaOH solution and passed over 10x100 cm columns
cont~inin~ a qll~t~rn~ry amlnonium substituted polystyrene resin. Resin would be f1rst washed
and pre-equilibrated to pH 7.0 with dilute buffer. Non-immunoglobulin proteins would be
absorbed under these conditions while the flow-through fraction would be enriched in
immlmnglobulins. In the ~lt~ i ve~ a process depicted as pathway B in Figure 1, and described
15 in Example 2, can be lltili~
The flow-through fraction would be concenkated by hollow fiber filtration (A/G
Technology), using polysulfone filtration c~ettes (30,000 MW cut offl. The resl-lting flow-
through concentrate would be centrifuged to remove excess non-polar lipids.
Rem;~ininp: phospholipids and residual non-Ig proteins would be precipitated by
20 sequential addition of the fiocculating agents chitosan (Pronova, Inc.) and caprylic acid. The
~cipiLaLion reaction would be carried-out using chromatographically deproteinized and defatted
whey at a temperature of 20-25 ~C. Chitosan would be added to a final concentration of 0.2%
and the pH of the mixture adjusted to pH 4.6. Caprylic acid would be added to a final
concentration of 5% by volume and the mixture stirred intermittently for 30 minllt~c
The resulting precipitate would be removed by centrifugation in a Sharples Centrifuge
(Alfa Laval, Model AS-16) and the supern~t~nt adjusted to pH6.5 by the addition of NaOH. The
centrifugation supernatant would be concentrated to approximately 20% solids using a hollow
fiber filtration system. After concentration, residual lactose, milk peptides and other salts would
be removed by step-wise diafiltration against three volumes of 15 mM potassium citrate pH 6.5.
The buffered immlm~-globulin fraction would be subsequently lyophilized to produce a
final powder. Antibodies to these antigens which are purified from whey by the procedures
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-26-
described can be incorporated into foods, drinks, tablets or capsules to prevent infection or
spread of H. pylori infections.
s-nlple 6 - Preparation of Clostridium Difficule Immuno~lobulins
This Example describes making an Ig product for preventing or treating Clostridium
tlifficllle infections. Cows would be immunized with inactive toxins A & B from C. difficile
together with other cell wall antigens that could promote aggregation or colonic bacterial levels.
Hypel; l l " "1 " ~e milk would be processed into provolone or mozzarella cheese by standard dairy
practices. The aqueous whey fraction co~ )g immunoglobulins would be clarified and
separated using standard dairy whey centrifugation methods. Clarified whey would be first
10 pasteurized by heating of 161~F for 15 sec. using a standard dairy HTST pasteurizer. The heat
treated whey would be concentrated sixfold (6X) using hollow fiber membranes with a molecular
weight cut off of 30,000 Daltons. Concentrated whey would be enriched in immunoglobulins by
anion exchange chromatography using an ISEP Chromatography System (Advanced Separation
Technologies) in a process generally depicted as pathway A of Figure 1. Whey concentrate in
this procedure would be first adjusted to pH 6.8 by addition of a NaOH solution and passed over
lOxlOO cm columns co~ ;llillp; a 4~ ",i.,y ammonium substituted polystyrene resin. Resin
would be first washed and pre-equilibrated to pH 7.0 with dilute buffer. Non-immunoglobulin
proteins would be absorbed under these conditions while the flow-through fraction would be
enriched in immllnoglobulins. In the alternative, a process depicted as pathway B of Figure 1,
20 and described in Example 2, can be lltili7t~(i
The flow-through fraction would be then concentrated by hollow fiber filtration (A/G
Technology), using polysulfone filtration cassettes (30,000 MW cut off). The resulting flow-
through concentrate would be centrifuged to remove excess non-polar lipids.
p~em~inin~ phospholipids and residual non-Ig proteins would be then precipitated by
25 sequential addition of the flocculating agents chitosan (Pronova, Inc.) and caprylic acid. The
precipitation reaction would be carried-out using chromatographically deproteinized and
tlef~tte~1 whey at a temperature of 20-25 ~C. Chitosan would be added to a final concentration of
0.2% and the pH of the ~ Lure adjusted to pH 4.6. Caprylic acid would be added to a final
concentration of 5% by volume and the mixture stirred interrnittently for 30 minutes.
The resulting precipitate would be removed by centrifugation in a Sharples Centrifuge
(Alfa Laval, Model AS-l 6) and the supern~t~nt adjusted to pH6.5 by the addition of NaOH. The
centrifugation sup~ ll would be concentrated to approximately 20% solids using a hollow
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fiber filtration system. After concentration, residual lactose, milk peptides and other salts would
be removed by step-wise diafiltration against three volumes of 15 mM potassium citrate pH 6.5.
The buffered immlmoglobulin fraction would be subsequently lyophili~cl to produce a
final powder. Antibodies to these antigens which are purified from whey by the procedures
5 described can be incorporated into colon specific deliverv formulations and ~lmini~t~red to
~lC~ colitis infections by C. difficile associated with prolonged oral ~r1mini~tration of
antibiotics.
FY~ ple 7 - Preparation of Vibrio cholerae Immuno~ bulins
This Example describes making an Ig product for ~l~v~ ing or treating Vibrio cholerae
10 infections. Cows would be illllllu~li~ed with inactivated cholera toxin (A &B subunit) or
individual subunits as well as cell antigens such as lipopolysacch~rides which are believed to
impart i~ ily to intt-stin~l infections. Hyperimmune milk would be processed into provolone
or mozzarella cheese by standard dairy practices. The aqueous whey fraction cO..~
immllnoglobulins would be clarified and separated using standard dairy whey centrifugation
methods. Clarified whey would be first pasteurized by heating of 1 61~F for 15 sec. using a
standard dairy HTST p~ . The heat treated whey would be concentrated sixfold (6X)
using hollow fiber membranes with a molecular weight cut off of 30,000 Daltons. Concentrated
whey would be enriched in immlln~globulins by anion exchange chromatography using an ISEP
Chromatography System (Advanced Separation Technologies) in a process generally depicted as
pathway A of Figure 1. Whey concentrate in this procedure would be first adjusted to pH 6.8 by
addition of a NaOH solution and passed over 10x100 cm columns c(~ ;-.i-.p a quaternary
ammonium substituted polystyrene resin. Resin would be first washed and pre-equilibrated to
pH 7.0 with dilute buffer. Non-imrnunoglobulin proteins would be absorbed under these
conditions while the flow-through fraction would be enriched in immlmt~globulins. In the
~1tPrn~tive, a process depicted as ~lhwdy B of Figure 1, and described in Example 2, can be
lltili7~-l
The flow-through fraction would be concentrated by hollow fiber filtration (A/G
Technology), using polysulfone filtration cassettes (30,000 MW cut off). The resllltin~ flow-
through conc~nLIdle would be centrifuged to remove excess non-polar lipids.
R~ g phospholipids and residual non-Ig proteins would be precipitated by
sequential ~ if io~l of the flocculating agents chitosan (Pronova, Inc.) and caprylic acid. The
precipitation reaction would be carried-out using chromatographically de~ eillized and defatted
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- -28-
whey at a temperature of 20-25 ~C. Chitosan would be added to a final concenkation of 0.2%
and the pH of the mixture adjusted to pH 4.6. Caprvlic acid would be added to a final
concelllld~ion of 5% by volume and the llli~lw~ stirred i~ ".~ ently for 30 min~ltes.
The resulting p~ le would be removed by centrifugation in a Sharples Centrifuge
5 (Alfa Laval, Model AS- 16) and the ~u~e~n~ l adjusted to pH6.5 by the addition of NaOH. The
centrifugation .,u~i "~ would be concentrated to approximately 20% solids using a hollow
fiber filtration system. After concentration, residual lactose, milk peptides and other salts would
be removed by step-wise diafiltration against three volumes of 15 mM potassium citrate pH 6.5.
The buffered imml-noglobulin fraction would be subsequently lyophili7~d to produce a
10 final powder. Antibodies to these antigens which are purified from whey by the procedures
described can be used in foods, drinks, or ~-imini~t~red as tablets or capsules to prevent oral
infections.
~n~ple 8
This Example describes making an immunoglobulin product wherein the cationic
15 polymer is a cationic fibrous cellulose. A cationic fibrous cellulose would be substituted for
chitosan in Examples 1-7 to precipitate phospholipids. A l,Lere,.ed cationic fibrous cellulose is
sold under the mark "DE-23" by Wh~tm~n, Inc. of New Jersey, USA.
Example ~
This Example describes making an immlmoglobulin product wherein the fatty acid has
20 the formula CH3 - (CH2)n ~ COOH wherein n is a whole integer from 4-5 and 7-10. Fatty acids,
where n is a whole integer from 4-5 and 7-10, would be substituted for caprylic acid in Example
1-7 to precipitate non-Ig proteins.
