WO1997012901A1 - 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

- 1 -

PROCESS FOR ISOLATING IMMUNOGLOBULINS IN WHEY

Field of the Invention

The present invention is directed to a process of isolating immunoglobulins from whey and whey concentrate, and a concentrated immunoglobulin product which is highly purified and readily administered. 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 immunoglobulin products effective for treatment of ETEC infection.

Background of the Invention Immunoglobulins or antibodies are made by higher animals in response to the presence of a foreign composition. Such a foreign composition, capable of eliciting an immure response, is referred to as an antigen. Immunoglobulins are complex proteins which are capable of specifically binding or attaching to the antigen.

Immunoglobulins play an important role in a host organism's fight against disease. Immunoglobulins, often abbreviated as lg, or antibodies abbreviated Ab, are made in several different forms. These classes of immunoglobulin are IgG, which is abundant in internal body fluids and certain lacteal secretions; IgA, abundant in sero-mucous secretions; IgM, an effective agglutinator; 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 immunoglobulin in lacteal secretions in humans. The level of antigen specific immunoglobulins present in milk or colostrum can be increased through parenteral or intra mammary immunization regimes.

Hyperimmune immunoglobulins derived from bovine milk or colostrum have been proposed for use in a variety of pharmaceutical/medicinal applications. Among these are oral and topical applications for the treatment or prevention 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 diarrhea.

Immunoglobulins for this purpose can be from colostrum, which is the first 4-5 milkings after calving, or from milk produced during the remainder 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 from colostrum is made difficult both by limited supplies and the complexities of collecting and processing 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 known that the majority of milk immunoglobulins pass into whey during conventional cheese making. Whey is a low cost and abundant byproduct ofthe cheese making industry and is readily available as a raw material for lg 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 presence of high concentrations (4-6 mg/ml) of non-immunoglobulin proteins including β- lactoglobulin and α-lactalbumin. Removal of greater than 90% of these proteins is required to produce a final product in which immunoglobulins constitute greater than 60% ofthe total protein.

Immunoglobulin products have been proposed for the treatment of ETEC in humans. 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 administration to subjects in the form of a food bar, reconstituted 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 formulation requires a highly concentrated 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. Summary 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 preparation that is greater than 60% by weight immunoglobulins. The method comprises forming an admixture ofthe 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 admixture wherein upon imposition of precipitation conditions the charged 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 admixture at a concentration wherein upon imposition of precipitation conditions the fatty acid forms a protein precipitate and a liquid phase. The method further comprises the step of imposing precipitation 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 immunoglobulins and can be further processed. Surprisingly and unexpectedly, the combination of a cationic polymer and a fatty acid allows simultaneous precipitation of non-immunoglobulin proteins and lipids to a degree that is greater than when the cationic polymer and fatty acid are used sequentially. The remaining eluant is >60% immunoglobulin. Such a purity is comparable with the concentration of immunoglobulins present in colostral whey and is comparable with the concentration and purity necessary for achieving a conveniently sized dose form.

Also surprisingly and unexpectedly, as described in greater detail below immunotherapy 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 treatment of active infection by pathogens in human subjects. The lipid precipitates and protein precipitates can be separated simultaneously by a relatively low speed centrifugation (6-12, 000 x g), to produce a clear supernatant having a high concentration of immunoglobulins. The supernatant can be further concentrated and diafiltered to produce a composition which is greater than 70% immunoglobulin protein and less than 0.1%) lipid by dry weight. This supernatant 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 comprising polypeptides and charged polysaccharides. A preferred charged polysaccharide is chitosan. 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 - 5.0. Preferably, in the formula:

CH₃ - (CH₂)_n - COOH n is 6; that is, the fatty acid preferably is caprylic acid. Caprylic acid forms colloid-like aggregates with non-immunoglobulin proteins at a pH of 4.5 - 5.0.

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 temperature 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 separate steps. Indeed, surprisingly and unexpectedly, the chitosan-lipid precipitate aids in the removal ofthe fatty acid-protein precipitate which by itself requires either lengthy high speed (> 15,000 x g) centrifugation or microfiltration for effective removal ofthe submicron size particulates. Only "low speed moderate time "or "high speed short time" centrifugation is necessary for the removal ofthe combined precipitates. This capability significantly facilitates large-scale manufacturing. In the event additional purity is desired, the coprecipitation of lipids and non lg proteins with chitosan and caprylic acid can be repeated.

Preferably, the lg rich supernatant is concentrated by ultrafiltration to remove low molecular weight protein and peptides, forming a further lg enriched retentate. Preferably, ultrafiltration 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 lg rich retentate is further concentrated by diafiltration to remove peptides, minerals and lactose, to form a dialyzed immunoglobulin concentrate. A preferred dialysis filtration buffer is a potassium citrate buffer of pH 6.5.

The immunoglobulin containing supernatant is preferably processed by sterile filtration. Sterile filtration is difficult with materials which have high lipid concentrations. The sterile filtrate is dried to form a dried immunoglobulin rich product.

Preferably, the dialyzed lg 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 mammal with one or more antigens that induce the production of antibodies against said one or more antigen, then collecting milk or colostrum from said mammal, and then processing 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 ofthe invention, the antigen is one or more colonization factor antigens.

Embodiments ofthe present invention are capable of using whey derived from pasteurized cheese whey. Cheese whey is pasteurized at 161 - 163°F for 15 to 17 seconds, or pasteurized for 30 minutes at 140°F - 142°F. In the alternative, the whey concentrate can be pasteurized.

Preferably, the concentrated whey ofthe first admixture is made by ultrafiltering pasteurized whey. Preferably, ultrafiltration is performed with a membrane having a 10,000- 150,000 Dalton molecular weight cut-off and, most preferably, a 30,000 Dalton molecular weight cut-off.

Concentrated whey ofthe first admixture 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 Daltons and, most preferably, 30,000 Daltons. A preferred hollow fiber ultrafiltration membrane is a polysulfone hollow fiber membrane. This membrane produces a concentration factor of 5-10 fold.

Preferably, the concentrated whey is further subjected to ion exchange chromatography to reduce the concentration of non-immunoglobulin proteins. A preferred chomotographic ion exchange process uses a strong anionic resin and whey protein concentrate having a pH of 6.5-

7.0. Anion exchange chromatography under these conditions can be used to remove from 20-

70% of non-immunoglobulin proteins from whey or whey protein concentrate without significantly altering immunoglobulin levels. Partially deproteinized whey or whey protein concentrates are a preferred starting material for whey immunoglobulin purification by the combined precipitation process described above. Because of reduced non-immunoglobulin protein levels and smaller processing volumes, reduced amounts of complexing agents are therefore required with this starting material.

Preferably, the antigen directed to ETEC comprises an antigen selected from the group consisting of Colonization Factor Antigens (CFA). A preferred group of CFAs comprise antigens of the CF A/I, CFA/II (CS 1 , CS2, CS3) and/or CF A/IV (CS4, CS5, CS6) families. The three most clinically prevalent families of CFA are identified in Table 1 below: TABLE 1 Colonization Factor Antigens of ETEC (CFAs)

Clinically Prevalent Families of CFAs

FAMILY ANTIGENS IN FAMILY

(1) CFA I CFA/I

(2) CFA II CS1 CS2 CS3 (present in all CFA/II strains)

(3) CFA/IV CS4 CS5 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 ofthe 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 preferred CFA-IV antigen is a CS6 antigen.

A further embodiment ofthe present invention features an immunoglobulin product derived from milk bearing mammals hyperimmunized with an antigen to produce immunoglobulins of interest. In one aspect ofthe invention the mammal is hyperimmunized 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 lg proteins. This product can be further processed to remove water to produce an lg product comprising at least 10% antibody, less than or equal to 6.0%o lipid and less than 20%) non-Ig protein which can be administered for the treatment of disease.

The immunoglobulin product also can comprise antibodies capable of binding antigens from any number of sources including antigens characteristic of other pathogenic organisms. Such pathogenic organism is preferably selected from one or more of the group consisting of Cryptosporidium parvum, Rotavirus, Shigellaflexneri, Heliobacter pylori, Clostridium difficile, Vibrio cholerae, Streptococcus mutans and Candida species Bacteriodes gingivalis, Bacteriodes melaninogenicus, Capnocytophaga species, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Streptococcus sobrinus and enterotoxigenic Escherichia coli.

According to another aspect ofthe invention, the immunoglobulin 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 hyperimmunizing bovines with a vaccine comprising isolated, CFA antigens preferably the majority ofthe antibodies in the immunoglobulin product that bind to Escherichia coli bind to colonization factor antigens of Escherichia coli. According to another aspect ofthe invention, a passive immunotherapy product is provided. It comprises immunoglobulins that bind to enterotoxigenic Escherichia coli, packaged in 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 infection in a human subject by enterotoxigenic Escherichia coli. Preferably the immunoglobulins bind colonization factor antigens as described above. In one embodiment the antigens are "purified". The term "purified", in the context ofthe present application, means substantially free of non-CFA antigens. That is, such non-CFA antigens, if present, are not sufficient to create an immune response more than two-fold over baseline, or unimmunized state. Preferred CFA antigens are CFA-I, CFA-II and CFA-III. The immunoglobulin product ofthe present invention can be administered to subjects as a reconstituted liquid, tablet, capsule, granules or food bar. Due to the removal of non- immunoglobulin proteins and lipids, an effective dose of immunoglobulin can be administered readily in a variety of formats. In one embodiment the effective dose is administered to an individual infected with enterotoxic Escherichia coli or at risk of being infected with the same, wherein the effective dose comprises antibodies that bind antigens of enterotoxigenic Escherichia coli, the antibodies formulated as a product containing at least 70% immunoglobulins, less than 6% lipid and less than 20%> non lg protein.

Embodiments ofthe present invention are capable of simultaneously removing the residual lipids, cheese culture bacteria, denatured protein aggregates and fatty acid precipitated whey protein. Lipid and protein precipitates can be removed by relatively low speed centrifugation in the presence ofthe chitosan. The simultaneous centrifugation ofthe protein precipitates with the lipid precipitates improved the recovery of highly purified immunoglobulins.

Protein and lipid precipitates are not readily removed by conventional microfiltration methods without also reducing the recovery of immunoglobulins because ofthe submicron size of much ofthe fatty acid protein precipitate. Although the concentration of lipid in separated whey is low, as whey is concentrated for processing, residual lipids reach levels of 5-20%) dry weight.

These high lipid concentrations compromise the processing as well as the storage of liquid lg products. Products with high levels of lipids cannot be sterile filtered and are subject to 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 apparent from the drawings and the detailed description which follows, which, by way of example, without limitation, describe preferred embodiments ofthe present invention.

Brief Description of the Drawings Fig. 1 depicts a flow diagram illustrating 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 ofthe 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 illustrates 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 ofthe whey comprising all or some of the whey proteins. "Partially deproteinized whey concentrate" refers to a part ofthe whey in which all or some ofthe nonimmunoglobulin proteins are removed. The present method can be used to make an immunoglobulin product capable of being administered in a relatively small dosage form. The process illustrated in Figure 1 begins with cheese making, 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 ofthe invention, the animals 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 CS1 and CS3 and, most preferably, CS3. A preferred CFA-IV is CS6. Animals immunized with such CFAs will secrete within their milk immunoglobulins that are directed to such antigens. Whey from one or more animals immunized with different antigens can be pooled to obtain immunoglobulins that are directed to a plurality of antigens. Typically, one would immunize all animals with all antigens.

Preferably, fat is separated from unconcentrated whey, obtained from cheese-making processes, using a standard dairy cream separator.

Preferably, the whey is concentrated by a factor of 5 to 10-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 temperature of 161 -163° F for a period of time of 15-17 seconds or 140 - 142° F for a period of time of 30 minutes. The whey can be pasteurized before or after concentration, using similar pasteurization conditions.

A preferred 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 temperature of 10-12°C. Concentrated whey produced by ultrafiltration may be subjected to an optional ion exchange 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 designated generally with the numeral 17. Following the anion exchange process, the flow-through comprises an immunoglobulin enriched fraction. Preferably, the immunoglobulins rich fraction is subjected to further ultrafiltration and further ion exchange chromatography to remove additional non-immunoglobulin proteins. The ultrafiltration step is designated 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 ofthe whey, then the lg fraction is centrifuged for fat removal after final ion exchange chromatography and ultrafiltration. The step of centrifugation can be performed on the concentrated whey product from an initial ultrafiltration step 15 as represented by pathway 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 intended 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., 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 5.0. 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 5.0. Thus, lipid precipitation conditions and protein precipitation conditions may be imposed simultaneously. Preferably, lipid precipitation conditions and protein precipitation conditions are imposed for 5 to 30 minutes after the admixture is formed by mixing. That is, a period of 5 to 30 minutes is allowed for the mixture to stand substantially motionless, with a 15 minute period preferred. An appropriate centrifuge capable of receiving the admixture containing a lipid 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 preferred bowl centrifuge is a Sharpies centrifuge and a preferred ejecting centrifuge is an automatically desludging Carr P-12 centrifuge or Alfa-Laval centrifuge. Preferably, the centrifuge assembly is maintained at a temperature of 20 to 25 °. Under these conditions the centrifuge separates the lipid precipitate and protein precipitate from the immunoglobulin rich supernatant. The lipid precipitate, comprising lipid and chitosan. aids in the removal ofthe protein precipitate allowing low gravity forces to remove substantially all of the colloidal particles. This precipitation step also substantially reduces bacterial contamination, primarily due to flocculation of contaminating microbes by chitosan. Following centrifugation the pH ofthe liquid supernatant 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 immunoglobulin rich supernatant 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 molecular weight cutoff of 10,000- 150,000 Daltons and most preferably 30,000 Daltons. This ultrafiltration step can produce a further concentration ofthe supernatant by 15 to 25-fold. The ultrafiltration allows further permeation of low molecular weight polypeptides through the membranes while retaining an immunoglobulin 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 maintained at a temperature of 10- 12 ° C .

The immunoglobulin rich retentate 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 utilizing a 15 mM potassium citrate buffer in demineralized tap water at a pH of 6.5. The diafiltration allows further permeation of 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. A final retentate from the diafiltration is dried by either freeze drying or spray drying. This step is generally designated by the numeral 33 in Figure 1. The dry powder is characterized as at least 85%o protein, which protein represents 70%> pure lg and less than 6%> lipid by weight.

A preferred method of administration comprises enteric coated tablets, capsules, or pellets. Individuals skilled in the art are able to formulate 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 each In 10,000

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

Eudragit™ 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 uniform 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 approximately 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 Eudragit™ 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. 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 hydroxypropyl methyl cellulose (HPMC) (Colorcon, West Point, PA) is a film coat. Eudragit™ L30D is an enteric polymer. Eudragit™ 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 dissolution and release ofthe immunoglobulin product at a pH of 5 or above.

These and other features will be apparent from the following examples which further highlight important aspects ofthe present invention.

Examples Example 1 - Production of ETEC antigens and preparation of anti-ETEC immunoglobulins

This Example features the making of an IgG product with activity against enterotoxigenic E. coli (ETEC). Materials and Methods Bacterial strains for vaccine production. Enterotoxigenic E. coli strains used in the manufacture of this product were obtained from the culture collection ofthe 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 M424C1 (CS1, CS3) and E9034A (CS3) (UM-Baltimore) were used for production of CS1 and CS3; strain HI 0407 (UM-Baltimore) was used for production of CFA-I. Strain M295 (W.R.A.I.R.), or CID553 (UT-Houston) is used for the production of CFA-IV.

Production of Bovine Vaccines

Expression of CFA/I. CS3. and CS6 from native organisms in broth cultures. Stock cultures were maintained in the frozen state at -80°C. Frozen cells were used to inoculate plates of CFA agar (lOg/L casamino acids, 6 g/L yeast extract, 50 mg/L magnesium sulfate, 5mg/L manganese chloride, 15 g/L agar) which were incubated ovemight 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 casamino acids, 6 g/L yeast extract, 50 mg/L magnesium sulfate, 5 mg/L manganese chloride) prewarmed to 37° C for fermentation. Preferably, sufficient quantities ofthe cell suspension are added to bring the optical density ofthe 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 m under 20-30 psi positive air pressure in a stainless steel, water-jacketed 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 - 1.2, 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 μm pore sized, low protein-binding membrane. Purification of CFA I. CS3. and CS6 from native organisms in broth cultures. CFAs were sheared from the surface ofthe 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 minutes. Ammonium sulfate was added to the CFA-rich supematant to 10-20%» saturation and incubated at 4°C with stirring for at least 30 minutes 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 supematant to 40-50% saturation and stirred at 4°C for at least 60 minutes followed by centrifugation preferably at 15,000-20,000 x g for 20-30 minutes to collect 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 size exclusion chromatography (CS3 or native CS6). Taking advantage ofthe large size of the CFA polymers, relatively pure CFAs elute in the void fraction in each case.

For CFA/I, radial flow chromatography is the method of choice using dimethyl 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 protein per 2-6 ml of resin is applied at a flow rate of 50-70 ml/min. Elution ofthe CFA-rich void fraction was observed by measuring the absorbance of the column effluent at 280 nm by standard methods. 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 containing a chaotropic agent, preferably N-lauryl sarcosine (10-40 mM), by standard methods. The resulting dialysate was then n 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 ofthe column effluent at 280 nm by standard methods. Expression of CS6 from recombinant organisms in broth cultures. Stock cultures were maintained in the frozen state at -80 °C. Frozen cells were used to inoculate plates of Luria Broth (LB) agar (10 g/L tryptone, 5 g/L yeast extract, 5 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 conferring resistance to a particular antibiotic. To ensure the stability and increase the copy number ofthe plasmid. 25-50 μg/ml ofthe appropriate 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, 5 g/L yeast extract, 5 g/L sodium chloride) containing 25 μg/ml ampicillin and prewarmed to 24-28 °C for fermentation. Preferably, sufficient quantities ofthe cell suspension are added to bring the optical density ofthe 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 φm under 5-10 psi positive air pressure in a stainless steel, water-jacketed 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 ofthe recombinant CS6 antigen is shed into the broth. The cells are removed, preferably, using tangential flow filtration with a 0.1 μm pore sized, low protein-binding membrane. The supematant 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 sufficient purity that no further purification is necessary. If purification is necessary, the size exclusion chromatography scheme outlined for CS3 above is utilized. Preparations 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 preparations were tested for bacterial and fungal sterility, and the presence of <100,000 EU/ml of endotoxin as determined by limulus lysate assay (Bio

Whittaker). The final vaccine was prepared by mixing the appropriate dose of antigen 1 :1 (v/v) with a synthetic non-LPS containing adjuvant. A preferred adjuvant is a Freund's adjuvant or its synthetic equivalent. Bovine vaccinations. All vaccinations were performed under USDA approval and administered under the direction of a licensed veterinarian. All animals used were healthy Holstein dairy cows. Health records were maintained and only healthy, mastitis-free animals were included in the study. A series of three intramuscular vaccinations were administered deep into the rear thigh muscle. A total volume of two ml was administered at a single site and the animals were monitored for adverse reaction. None were observed. Vaccinations were given three weeks apart, and milk collected regularly beginning one week after the third shot. Milk samples were taken from every batch and shipped frozen to ImmuCell coφoration (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 immunoglobulin (AEMI). To simulate continuous production, milk from seven different batches, collected over a four week period, were pooled to make the clinical test material described herein.

Vaccinations were performed separately with CSL CS3 and CS6, alone and in combination with equally successful results.

Preparation of anti-E. coli Bovine Milk Immunoglobulin. Hyperimmune milk was processed into provolone or mozzarella cheese by standard dairy practices. The aqueous whey fraction containing immunoglobulins was clarified and separated using standard dairy whey centrifugation methods. Clarified whey was first pasteurized by heating of 159°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 ISΕP 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 10x100 cm columns containing a quaternary ammonium substituted polystyrene resin. Resin was first washed and pre-equilibrated to pH 7.0 with dilute buffer. Non-immunoglobulin proteins were absorbed under these conditions while the flow-through fraction was enriched in immunoglobulins. The flow-through fraction was then concentrated by hollow fiber filtration (A/G Technology Coφ., Needham, MA), using polysulfone filtration cartridges (30,000 MW cut off, 24m² surface area). The resulting flow-through concentrate was centrifuged to remove excess non-polar lipids.

Remaining phospholipids and residual non-Ig proteins were then precipitated by sequential addition ofthe flocculating agents chitosan (Sea Cure 443, Pronova Biopolymers, 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 ofthe 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 minutes static incubation. The resulting precipitate was removed by centrifugation in a Shaφles Centrifuge (Model

AS- 16, Alfa-Laval, Warminster, PA) and the supematant adjusted to pH6.5 by the addition of NaOH. The centrifugation supematant 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 6.5.

The buffered immunoglobulin fraction was subsequently lyophilized to produce a final powder. Analysis of a representative lot of anti-E coli immunoglobulin produced by this procedure revealed that the lyophilized powder contained 78%) protein, 5.5%o fat, 1.1% carbohydrate, 10.5% ash due to added potassium citrate buffer, 2.2%> residual ash and 2.7% moisture. Ig comprised 79% ofthe total protein as revealed by scanning densitometry and SDS- polyacrylamide gel electrophoresis. Additional milk proteins present included beta- lactoglobulin, alpha-lactalbumin, serum albumin, and trace amounts of casein. Table 1 below describes the recovery of immunoglobulin activity and the Ig purity at different stages in this purification process. TABLE 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 Flow Through 108 783 79.8% 8.85%

Defatted Concentrate 1861 45.6 79.9% 11%

Ig Concentrate 4750 1 1.4 51.1% 79%o

Ouantitation of anti-CFA activitv in ETEC Directed Product bv ELISA. Anti-CFA titers 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 preparation used to coat the wells. Thus, the most accurate and meaningful comparison of multiple samples was made by establishing a reference standard from which all unknown samples titers were inteφolated. Dilutions of our anti-CFA milk standard were run on each plate containing unknown samples and a standard curve was constmcted.

Titers for unknown samples were then inteφolated from the standard curve. This normalized all ELISA data and permitted meaningful comparisons between samples n on different assays to be made.

The ETEC product ofthe present invention derived from bovines hyperimmunized with purified CFAs exhibited titers which were 3-10 fold higher than titers derived from products derived from bovines immunized with whole cell extracts. These results are set forth in Table 2 below: TABLE 2

1 2 3

Vaccine Fold Increase Ratio (Ag/WC)

1 Whole Cell 12.2 1

2 CFA/I 82.8 6.8

3 CS3 42.4 3.5

4 CS6 46 3.8

5 CS1 61.1 5.1

Volunteer study. Twenty-five healthy adult volunteers, housed as in-patients in the isolation ward at the Center for Vaccine Development (University of Maryland School of Medicine), were randomly assigned to three groups. Placebo (n=10), High dose (n=l 1), and Low dose (n=4) by assigning subject ID numbers to identically packaged foil pouches containing measured doses of each test article. All investigators and volunteers were blinded to these treatment group assignments throughout the study and during assessment of outcome. Each placebo pouch contained 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 ofthe two doses of product after meals for two days. Each dose consisted ofthe assigned pouch of product as a powder dissolved in 8 ounces (240 ml) of water containing 2g of sodium bicarbonate. On day three, two hours after consuming the morning dose, all volunteers drank 4 ounces (120 ml) of water containing 2g of sodium bicarbonate. One minute later, each received an oral challenge inoculum containing IO⁹ of H10407 (O78:Hl l), a CFA/I-bearing ETEC strain suspended in 1 ounce (30 ml) of water containing 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 administered 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 nurse. Daily stool samples were taken for bacteriology examination.

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 course of ciprofloaxcin (500 mg b.i.d.) to eradicate the challenge organism. The primary effectiveness variable was the clinical diagnosis of diarrhea, 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. Clinical. Seven out ofthe 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 Hyperimmune Immunoglobulin in Healthy Normal Volunteers". The primary effectiveness variable defined for the study described above was the clinical diagnosis of diarrhea 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 represented by bars with small dots with fine spacing. Each bar is separately labeled for placebo, high does and low dose. Comparing the attack rate in the placebo group (70%) with the attack rate in the treated groups (6.7%>), prophylactic administration of ETEC product brought about a 90%> protection rate. The mean stool volume in volunteers with diarrhea 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 diaπhea, 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 cramping symptoms are depicted with bars with light cross hatching. Patients exhibiting symptoms of gurgling are depicted with bars with dark cross hatching.

Bacteriology. Daily stool samples were analyzed for the presence of the challenge organism to quantitate shedding over time. The average number of challenge organisms per gram of stool shed by volunteers receiving the placebo at the time of maximal shedding was 4.5 x 10⁸ CFU/g. The peak mean value for volunteers receiving the ETEC product was 6.2 x 10⁷/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, immunoglobulin 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 altemative to existing dmg interventions.

Example 2 - Preparation of Anti-Cryptosporidium parvum Immunoglobulins

Hyperimmune milk from cows immunized with a killed C. parvum vaccine was processed into provolone or mozzarella cheeses by standard cheese making procedures. The aqueous whey fraction containing immunoglobulins was clarified and separated using standard whey centrifugation methods. The schematic for anύ-Cryprosporidium immunoglobulin purification in this example is shown in Figure 1.

Using pasteurization and hollow fiber ultrafiltration procedures described for initial immune whey processing in Example 1, a 6X whey concentrate was prepared and subjected to direct chitosan/caprylic treatment as outlined in Figure 1 (pathway B). Chitosan was added to a 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 intermittent stirring.

The chitosan-lipid and caprylic-protein precipitates were separated by centrifugation in a Sorvall centrifuge at 10,000 x g and the resulting supematant 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. - 22 -

Ig purity at different steps was determined by densitometric scanning of 4-20% SDS- PAGE gels mn under non-reducing conditions at pH 8.5 which were stained with Coomassie Blue. The results of this purification are shown in Table 2 below.

TABLE 2 Summary of Anti-Cryptosporidium parvumlmmunog\obulm Purification

Anti- % % Crypto Antibody Purity

Process Activity Activity Ig/Total Intermediate (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 Supematant 3476 75.5 66.7

Example 3- Preparation of Rotavirus Immunoglobulins

This Example describes making an Ig product for preventing or treating Rotavims infections. Cows would be immunized with a vaccine containing killed vims or purified viral neutralization antigens (eg. G or P antigens) representing the four major rotavims types (1-4) infective for humans. Hyperimmune milk would be processed into provolone or mozzarella cheese by standard dairy practices. The aqueous whey fraction containing immunoglobulins 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 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 10x100 cm columns containing a quaternary 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 immunoglobulins. In the altemative, a process depicted in pathway B of Figure 1 , and described in Example 2 can be utilized.

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. Remaining phospholipids and residual non-Ig proteins would be precipitated by sequential addition ofthe 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 ofthe 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 minutes.

The resulting precipitate would be removed by centrifugation in a Shaφles Centrifuge (Alfa Laval, Model AS- 16) and the supematant adjusted to pH6.5 by the addition of NaOH. The centrifugation supematant 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 immunoglobulin fraction would be subsequently lyophilized to produce a final powder. Such antibodies purified from whey by the procedures described can be incoφorated into foods or drinks to prevent rotavims infections in young children and older adults. Example 4 - Preparation of Shigella flexneri Immunoglobulin

This Example describes making an Ig product for preventing or treating Shigella flexneri infections. Cows are immunized with a vaccine containing 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 aqueous whey fraction containing immunoglobulins 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. 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 10x100 cm columns containing a quaternary 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 was enriched in immunoglobulins. In the altemative, pathway B - a process depicted as described in Figure 1 , and Example 2, can be utilized.

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.

Remaining phospholipids and residual non-Ig proteins would be precipitated by sequential addition ofthe 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 ofthe 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 minutes. The resulting precipitate would be removed by centrifugation in a Shaφles Centrifuge

(Alfa Laval, Model AS- 16) and the supematant adjusted to pH6.5 by the addition of NaOH. The centrifugation supematant 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 immunoglobulin fraction would be subsequently lyophilized to produce a final powder. Antibodies to these antigens which are present in whey can be purified by the procedures described and administered in food, drink or capsule/tabled from for the prevention of Shigella infections among susceptible or exposed individuals. Example 5 - Preparation of Heliobacter pylori Immunoglobulins This Example describes making an Ig product for preventing or treating Heliobacter pylori infections. Cows would be immunized with purified antigens of H.pylori represented by presumed virulence factors such as urease. vacuolating cytoxins and flagella which are thought to be important in bacterial infection of gastric mucosa. Hyperimmune milk would be processed into provolone or mozzarella cheese by standard dairy practices. The aqueous whey fraction containing immunoglobulins 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 immunoglobulins by anion exchange chromatography using an I SEP 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 containing a quaternary 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 immunoglobulins. In the altemative, a process depicted as pathway B in Figure 1, and described in Example 2, can be utilized.

The flow-through fraction would be 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.

Remaining phospholipids and residual non-Ig proteins would be precipitated by sequential addition ofthe 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 ofthe 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 minutes. The resulting precipitate would be removed by centrifugation in a Shaφles Centrifuge

(Alfa Laval, Model AS-16) and the supematant adjusted to pH6.5 by the addition of NaOH. The centrifugation supematant 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 immunoglobulin fraction would be subsequently lyophilized to produce a fmal powder. Antibodies to these antigens which are purified from whey by the procedures described can be incoφorated into foods, drinks, tablets or capsules to prevent infection or spread of H. pylori infections.

Example 6 - Preparation of Clostridium Difficule Immunoglobulins

This Example describes making an Ig product for preventing or treating Clostridium difficule 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. Hyperimmune milk would be processed into provolone or mozzarella cheese by standard dairy practices. The aqueous whey fraction containing immunoglobulins 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 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 10x100 cm columns containing a quaternary 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 immunoglobulins. In the altemative, a process depicted as pathway B of Figure 1, and described in Example 2, can be utilized.

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.

Remaining phospholipids and residual non-Ig proteins would be then precipitated by sequential addition ofthe 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 ofthe 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 minutes. The resulting precipitate would be removed by centrifugation in a Shaφles Centrifuge

(Alfa Laval, Model AS-16) and the supematant adjusted to pH6.5 by the addition of NaOH. The centrifugation supematant 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 immunoglobulin fraction would be subsequently lyophilized to produce a final powder. Antibodies to these antigens which are purified from whey by the procedures described can be incoφorated into colon specific delivery formulations and administered to prevent colitis infections by C. difficile associated with prolonged oral administration of antibiotics. Example 7 - Preparation of Vibrio cholerae Immunoglobulins

This Example describes making an Ig product for preventing or treating Vibrio cholerae infections. Cows would be immunized with inactivated cholera toxin (A &B subunit) or individual subunits as well as cell antigens such as lipopolysaccharides which are believed to impart immunity to intestinal infections. Hyperimmune milk would be processed into provolone or mozzarella cheese by standard dairy practices. The aqueous whey fraction containing immunoglobulins 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 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 10x100 cm columns containing a quaternary 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 immunoglobulins. In the altemative, a process depicted as pathway B of Figure 1 , and described in Example 2, can be utilized.

The flow-through fraction would be 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. Remaining phospholipids and residual non-Ig proteins would be precipitated by sequential addition ofthe 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 ofthe 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 minutes.

The resulting precipitate would be removed by centrifugation in a Shaφles Centrifuge (Alfa Laval, Model AS- 16) and the supematant adjusted to pH6.5 by the addition of NaOH. The centrifugation supematant 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 immunoglobulin fraction would be subsequently lyophilized to produce a final powder. Antibodies to these antigens which are purified from whey by the procedures described can be used in foods, drinks, or administered as tablets or capsules to prevent oral infections.

Example 8

This Example describes making an immunoglobulin product wherein the cationic polymer is a cationic fibrous cellulose. A cationic fibrous cellulose would be substituted for chitosan in Examples 1 -7 to precipitate phospholipids. A preferred cationic fibrous cellulose is sold under the mark "DE-23" by Whatman, Inc. of New Jersey, USA.

Example 9

This Example describes making an immunoglobulin product wherein the fatty acid has 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

CLAIMS:

1. A method of isolating immunoglobulins, comprising the steps:

(a) providing a first admixture of whey, whey concentrate, whey fractions, or partially deproteinized whey concentrate and a cationic polymer and a fatty acid, wherein said cationic polymer has a concentration in said admixture to form a lipid-polymer precipitate upon imposition of precipitation conditions and wherein said fatty acid has a concentration in said admixture to form a protein precipitate upon imposition of said precipitation conditions,

(b) imposing said precipitation conditions on said admixture to form a immunoglobulin rich supematant and precipitate which includes both a protein precipitate and a lipid-polymer precipitate, and

(c) separating said immunoglobulin rich supematant from said precipitate.

2. The method of claim 1 , further comprising the steps of:

(a) first vaccinating a milk bearing mammal with one or more antigens, said antigen inducing the production of Ig that binds to said antigen,

(b) collecting milk or colostrum from said mammal and processing said milk or colostrum to form whey, whey concentrate, whey fractions, or partially deproteinized whey concentrate, prior to providing the first admixture.

3. The method of claim 1 , wherein the immunoglobulin rich supematant contains immunoglobulins capable of binding one or more antigens associated with enterotoxigenic Escherichia coli.

4. The method of claim 3 wherein said one or more antigens are selected from the group consisting of CFA-I, CFA-II and CFA-IV antigens.

5. The method of claim 3 wherein said one or more antigens comprise an antigen from each ofthe groups consisting of CFA-I, CFA-II and CFA-IV.

6. The method of claim 3 wherein said CFA-II antigen comprises at least one antigen selected from the group consisting of CS1 and CS3.

The method of claim 3 wherein said CFA-IV antigen is CS6.

8. The method of claims 1, 2, 3, 4, 5, 6 or 7 wherein said cationic polymer is selected from the group comprising polypeptides and polysaccharides.

9. The method of claims 1, 2, 3, 4, 5, 6, or 7 wherein said cationic polymer is chitosan.

10. The method of claims 1, 2, 3, 4, 5, 6 or 7 wherein said fatty acid has the formula CH₃ - (CH₂)_n - COOH wherein n is a whole integer from 4-10.

11. The method of claims 1 , 2, 3, 4, 5, 6, or 7 wherein said fatty acid is caprylic acid.

12. The method of claims 1, 2, 3, 4, 5, 6 or 7 wherein said cationic polymer is chitosan and wherein said fatty acid is caprylic acid.

13. The method of claim 1 wherein said protein and lipid precipitates are separated from said immunoglobulin supematant by centrifugation.

14. The method of claims 1 or 13 wherein said immunoglobulin rich supematant is concentrated by ultrafiltration with a membrane having a molecular weight cutoff of 10,000 - 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 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 infection in a human subject by enterotoxigenic Escherichia coli.