CA2233935A1 - Process for isolating immunoglobulins in whey - Google Patents

Abstract

The present invention is directed to a process of isolating immunoglobulin from whey or whey concentrate and a concentrated immunoglobulin product which is highly purified. The process features the co-precipitation lipids and non-immunoglobulin proteins simultaneously with a charged polymer and a fatty acid.

Description

CA 0223393~ 1998-04-02 W O 97/12901 PCT~US96/1~945 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 CA 0223393~ 1998-04-02 W O 97/12901 PCT~US96/15945.. -2-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 CA 0223393~ 1998-04-02 W ~ 97/12901 PCTAUS96/15945 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

CA 0223393~ 1998-04-02 W O 97/12901 PCT~US96/15945 . --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 CA 0223393~ 1998-04-02 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:

CA 0223393~ 1998-04-02 W O 97/12901 PCT~US96/15945 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 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 CA 0223393~ 1998-04-02 W O 97/12901 PCT~US96/15945 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 CA 0223393~ 1998-04-02 W O 97/12901 PCT~US96/15945.. -8-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.

-CA 0223393~ 1998-04-02 W O 97/12901 PCTrUS96/15945 . _ 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 CA 0223393~ 1998-04-02 W O 97/12901 PCT~US96/~5945 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.
-CA 0223393~ 1998-04-02 '- - 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.

CA 0223393~ 1998-04-02 W O 97/12901 PCT~US96/15945 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.

W O 97/12901 PCT~US96/15945 -13-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 CA 0223393~ 1998-04-02 W ~ 97/12901 PCTrUS96/15945 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.

CA 0223393~ 1998-04-02 W O 97/12901 PCT~US96/15945 '- - 15 -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~

CA 0223393~ 1998-04-02 W O 97/12901 PCT~US96/15945 -16-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 CA 0223393~ 1998-04-02 W O 97/12901 PCT~US96/15945 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.

W O 97/12901 PCT~US96/15945 '- -18-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:

CA 0223393~ 1998-04-02 W O 97/12901 PCT~US96/1594 . - 19-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 CA 0223393~ 1998-04-02 W~ 97/12901 PCTAUS96/15945 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 CA 0223393~ 1998-04-02 W O 97/12901 PCT~US96/15945 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.

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.

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 CA 0223393~ l998-04-02 W O 97/12901 PCT~US96/15945 -23-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 CA 0223393~ 1998-04-02 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 CA 0223393~ 1998-04-02 W O 97/12901 PCT~US96/1594 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 CA 0223393~ 1998-04-02 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 W O 97/12901 PCT~US96/15945- -27-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 W O 97/12901 PCTnUS96/15945 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.

Claims (35)

1. A method of isolating immunoglobulin from whey, concentrated whey or whey fractions derived from a milk-bearing mammal, the method comprising the following steps in order:
a) providing a sample comprising whey, whey concentrate or whey fractions;
b) forming an admixture comprising the sample of step a) and a cationic polymer, the concentration of the cationic polymer in the admixture being appropriate for lipid precipitation;
c) forming a second admixture comprising the admixture of step b) and a fatty acid, the concentration of the fatty acid in the second admixture being appropriate for precipitation of non- immunoglobulin proteins;
d) precipitating lipids and non-immunoglobulin proteins from the second admixture of step c) to form an immunoglobulin-rich supernatant and a lipid and non-immunoglobulin protein precipitate; and e) isolating the immunoglobulin-rich supernatant from the mixture of step d).

2. The method of Claim 1 wherein the milk-bearing mammal is a cow or a goat.

3. The method of Claim 1 wherein the milk-bearing mammal is pre-immunized with an antigen to stimulate production of immunoglobulin which binds specifically to the antigen.

4. The method of Claim 3 wherein the antigen is associated with enterotoxigenic Escherichia coli.

5. The method of Claim 3 wherein said antigen is selected from the group consisting of CFA-I, CFA-II and CFA-IV antigens.

6. The method of Claim 3 wherein the milk-bearing mammal is pre-immunized with an antigen from each of the groups comprising CFA-I, CFA-II and CFA-IV antigens.

7. The method of Claim 5 wherein said CFA-II antigen comprises at least one antigen selected from the group consisting of CS1 and CS3.

8. The method of Claim 5 wherein said CF-IV antigen is CS6.

9. The method of Claim 1 wherein said cationic polymer is selected from the group consisting of polypeptides and polysaccharides.

10. The method of Claim 9 wherein said cationic polymer is chitosan.

11. The method of Claim 1 wherein said fatty acid has the formula CH3-(CH2)n-COOH wherein n is a whole integer from 4-10.

12. The method of Claim 12 wherein said fatty acid is caprylic acid.

13. The method of Claim 1 wherein said lipid and non-immunoglobulin protein precipitate are separated from said immunoglobulin-rich supernatant by centrifugation.

14. The method of Claim 13 further comprising concentrating the immunoglobulin-rich supernatant by ultrafiltration with a membrane having a molacular weight cutoff of from about 20,000 to about 150,000 Daltons to remove polypeptides and lactose to form a retentate.

15. The method of Claim 14 wherein said retentate is diafiltered to form a dialyzed immunoglobulin concentrate.

16. The method of Claim 15 wherein said dialyzed immunoglobulin concentrate is freeze-dried or spray-dried to form a powder.

17. A method for isolating immunoglobulin from whey, concentrated whey or whey fractions derived from a milk-bearing mammal, the method comprising the following steps in order:
a) providing a sample comprising whey, whey concentrate or whey fractions;
b) forming an admixture comprising the sample of step a) and chitosan, the concentration of the chitosan in the admixture being appropriate for lipid precipitation;
c) forming a second admixture comprising the admixture of step b) and caprylic acid, the concentration of the caprylic acid in the second admixture being appropriate for precipitation of non-immunoglobulin proteins;
d) precipitating lipids and non-immunoglobulin proteins from the second admixture of step c) to form an immunoglobulin-rich supernatant and a lipid and non-immunoglobulin protein precipitate; and e) isolating the immunoglobulin-rich supernatant from the mixture of step d).

18. The method of Claim 17 wherein the milk-bearing mammal is a cow or a goat.

19. The method of Claim 17 wherein the milk-bearing mammal is pre-immunized with an antigen to stimulate production of immunoglobulin which binds specifically to the antigen.

20. The method of Claim 19 wherein the antigen is associated with enterotoxigenic Escherichia coli.

21. The method of Claim 20 wherein said antigen is selected from the group consisting of CFA-I, CFA-II and CFA-IV antigens.

22. The method of Claim 20 wherein the milk-bearing mammal is pre-immunized with an antigen from each of the groups comprising CFA-I, CFA-II and CFA-IV antigens

23. The method of Claim 21 wherein said CFA-II antigen comprises at least one antigen selected from the group consisting of CS1 and CS3.

24. The method of Claim 21 wherein said CF-IV antigen is CS6.

25. A passive immunotherapy product comprising immunoglobulins that bind to enterotoxigenic Escherichia coli, packaged in a unit dosage form of 1 gram or less and present in an amount effective for treating active infection in a human subject by or for prophylaxis of infections in a human subject by enterotoxigenic Escherichia coli.

26. The product of claim 25 wherein said immunoglobulins bind colonization factor antigens.

27. The product of claim 26 wherein the colonization factor immunoglobulins are selected from the group consisting of CFA-I, CFA-II and CFA-IV.

28. The product of claim 27 wherein said immunoglobulins collectively bind all CFA antigens from the groupe CFA-I, CFA-II and CFA-IV.

29. The product of claim 26 wherein the immunoglobulins bind to CS3.

30. The product of claim 26 wherein the immunoglobulins bind to CS6.

31. The product of claim 25 wherein said immunoglobulin are enteric coated.

32. An immunoglobulin product isolated from milk or colostrum of a milk bearing mammal comprising antibodies that bind to enterotoxic Escherichia coli, and wherein said antibodies exhibit a range of 3-50 fold higher titer than titers achieved by inoculation of bovines with a whole cell extract of enterotoxigenic Escherichia coli.

33. The immunoglobulin product of claim 32 wherein the majority of the antibodies in the immunoglobulin product that bind to Escherichia coli bind to colonization factor antigens of Escherichia coli.

34. A method of treating an individual infected with enterotoxigenic Escherichia coli or at risk of being infected with enterotoxigenic Escherichia coli, comprising the step of administering an effective dose of a product comprising antibodies that bind antigens associated with enterotoxigenic Escherichia coli comprising at least 70%
immunoglobulins, less than 6% lipid, and less than 20%
non-Ig protein.

35. A vaccine for hyperimmunizing milk producing animals comprising isolated antigens, said antigens selected from each of the colonization factor antigens (CFAs) consisting of CFA-I, CFA-II and CFA-IV.