EP0859783A1 - Procede d'isolement d'immunoglobuline a partir de petit lait - Google Patents

Procede d'isolement d'immunoglobuline a partir de petit lait

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Publication number
EP0859783A1
EP0859783A1 EP96934050A EP96934050A EP0859783A1 EP 0859783 A1 EP0859783 A1 EP 0859783A1 EP 96934050 A EP96934050 A EP 96934050A EP 96934050 A EP96934050 A EP 96934050A EP 0859783 A1 EP0859783 A1 EP 0859783A1
Authority
EP
European Patent Office
Prior art keywords
whey
cfa
immunoglobulin
immunoglobulins
antigens
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96934050A
Other languages
German (de)
English (en)
Inventor
Daniel J. Freedman
Joseph H. Crabb
Frank E. Ruch
Elizabeth A. Acker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Immucell Corp
Original Assignee
Immucell Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/539,539 external-priority patent/US5747031A/en
Application filed by Immucell Corp filed Critical Immucell Corp
Publication of EP0859783A1 publication Critical patent/EP0859783A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/04—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from milk
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/121—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Helicobacter (Campylobacter) (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K16/1232—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia from Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1239—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Vibrionaceae (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • ETEC enterotoxic E. coli
  • 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.
  • ETEC Enterotoxigenic E. coli
  • 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.
  • 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 ⁇ - lactoglobulin and ⁇ -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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • the fatty acid and cationic charged polymer are selected to have precipitation conditions which are similar.
  • 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.
  • CH 3 - (CH 2 ) 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.
  • 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.
  • 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.
  • 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.
  • the lg rich supernatant is concentrated by ultrafiltration to remove low molecular weight protein and peptides, forming a further lg enriched retentate.
  • 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.
  • 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.
  • 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.
  • 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.
  • the antigen is characteristic of enterotoxic Escherichia coli.
  • 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.
  • the whey concentrate can be pasteurized.
  • the concentrated whey ofthe first admixture is made by ultrafiltering pasteurized whey.
  • 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.
  • 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.
  • 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-
  • the antigen directed to ETEC comprises an antigen selected from the group consisting of Colonization Factor Antigens (CFA).
  • CFA Colonization Factor Antigens
  • 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)
  • CFA/III one member
  • CS17 CS17
  • PCF0159 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.
  • 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.
  • 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.
  • 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.
  • a passive immunotherapy product is provided.
  • 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.
  • the immunoglobulins bind colonization factor antigens as described above.
  • the antigens are "purified".
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • fat is separated from unconcentrated whey, obtained from cheese-making processes, using a standard dairy cream separator.
  • the whey is concentrated by a factor of 5 to 10-fold over whey recovered directly from cheese making processes.
  • 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.
  • the whey concentrate is pasteurized.
  • 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.
  • 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.
  • 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.
  • the flow-through comprises an immunoglobulin enriched fraction.
  • 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.
  • 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.
  • 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.
  • lipid precipitation conditions and protein precipitation conditions may be imposed simultaneously.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • enteric formulations There are many possible enteric formulations. One formulation is set forth below:
  • 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.
  • HPMC hydroxypropyl methyl cellulose
  • 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.
  • the milk protein would comprise a purified IgG product.
  • Avicel PH101 FMC Corp., Newark, DE
  • Avicel PH101 is a binder.
  • Avicel PH101 is a microcrystalline cellulose.
  • Other binders may be substituted for microcrystalline cellulose.
  • Ac-Di-Sol FMC Corp., Newark, DE
  • Ac-Di-Sol is a disintegrant.
  • Sol is a cross-linked sodium carboxymethylcellulose.
  • Other disintegrants may be substituted for Ac-Di-Sol 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.
  • hydroxypropyl methyl cellulose (HPMC) (Colorcon, West 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.
  • the coatings, polymers and plasticizers allow the granules to survive gastric acid for two hours.
  • the coatings, polymers and plasticizers allow dissolution and release ofthe immunoglobulin product at a pH of 5 or above.
  • This Example features the making of an IgG product with activity against enterotoxigenic E. coli (ETEC).
  • ETEC enterotoxigenic E. coli
  • 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.
  • 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 ).
  • Cells were harvested and concentrated 40-50 fold, preferably using tangential flow filtration with a 0.1 ⁇ m 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
  • 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.
  • radial flow chromatography is the method of choice using dimethyl amino ethyl substituted (DEAE) cellulose, preferably cross-linked for support.
  • DEAE dimethyl amino ethyl substituted
  • 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.
  • 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.
  • CS6 Fusion 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.
  • LB Luria Broth
  • yeast extract 5 g/L yeast extract, 5 g L sodium chloride, 15 g L agar
  • the genes encoding the recombinant CFA are found on an extrachromosomal element (plasmid) which also expresses proteins conferring resistance to a particular antibiotic.
  • plasmid extrachromosomal element
  • 25-50 ⁇ g/ml ofthe appropriate antibiotic were included in all growth media.
  • the culture was aerated, preferably by mixing at 40-60 ⁇ m under 5-10 psi positive air pressure in a stainless steel, water-jacketed fermenter.
  • 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 ⁇ m 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)
  • 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.
  • Vaccinations were performed separately with CSL CS3 and CS6, alone and in combination with equally successful results.
  • Concentrated whey was enriched in immunoglobulins by anion exchange chromatography using an IS ⁇ P 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 Co ⁇ ., Needham, MA), using polysulfone filtration cartridges (30,000 MW cut off, 24m 2 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 Sha ⁇ les Centrifuge (Model
  • 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.
  • 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 inte ⁇ olated. Dilutions of our anti-CFA milk standard were run on each plate containing unknown samples and a standard curve was constmcted.
  • 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.
  • 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 an ⁇ -Cryprosporidium immunoglobulin purification in this example is shown in Figure 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.
  • 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.
  • 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
  • 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 Sha ⁇ les 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.
  • 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 inco ⁇ orated into foods or drinks to prevent rotavims infections in young children and older adults.
  • 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.
  • 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 Sha ⁇ les Centrifuge
  • 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.
  • 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.
  • 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 Sha ⁇ les Centrifuge
  • 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.
  • 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 Sha ⁇ les Centrifuge
  • 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 inco ⁇ orated 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 Sha ⁇ les 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.
  • 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.
  • 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.
  • This Example describes making an immunoglobulin product wherein the fatty acid has the formula CH 3 - (CH 2 ) 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

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Abstract

L'invention porte sur procédé d'isolement d'immunoglobuline à partir de petit lait ou de petit lait concentré et sur un concentré d'immunoglobuline hautement purifié. Ledit procédé consiste en une co-précipitation des lipides et des protéines différentes de l'immunoglobuline, et d'un polymère portant des charges et d'un acide gras.
EP96934050A 1995-10-05 1996-10-04 Procede d'isolement d'immunoglobuline a partir de petit lait Withdrawn EP0859783A1 (fr)

Applications Claiming Priority (5)

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US539539 1995-10-05
US08/539,539 US5747031A (en) 1995-10-05 1995-10-05 Process for isolating immunoglobulins in whey
US62327696A 1996-03-28 1996-03-28
US623276 1996-03-28
PCT/US1996/015945 WO1997012901A1 (fr) 1995-10-05 1996-10-04 Procede d'isolement d'immunoglobuline a partir de petit lait

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WO2003055502A1 (fr) * 2001-12-24 2003-07-10 Fonterra Co-Operative Group Limited Composition d'immunoglobuline
EP1623717B1 (fr) * 2003-03-14 2018-08-29 Meiji Co., Ltd. Composition contre l' infection à rotavirus et son procédé de production
WO2006127798A2 (fr) * 2005-05-24 2006-11-30 The United States Of America As Represented By The Secretary Of The Navy Produit immunoprophylactique passif a base d'anti-adhesine
WO2010151632A1 (fr) * 2009-06-25 2010-12-29 Bristol-Myers Squibb Company Purification de protéines par précipitation de l'acide caprylique (l'acide octanoïque)
JP5985643B2 (ja) 2011-09-12 2016-09-06 スカンジナビアン バイオファーマ ホールディング アーベー 細胞表面上におけるｅｔｅｃｃｓ６抗原の提示を増大させるための方法およびその得られる産物
US10738078B2 (en) 2014-11-03 2020-08-11 Bristol-Myers Squibb Company Use of caprylic acid precipitation for protein purification
FR3076294B1 (fr) 2017-12-29 2022-01-28 Lab Francais Du Fractionnement Procede de purification d'anticorps a partir de lait brut
WO2020053661A1 (fr) * 2018-09-13 2020-03-19 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Procédés de purification d'anticorps émanant du lait de mammifères non humains transgéniques comprenant l'utilisation de chitosane
CN111718410B (zh) * 2020-06-05 2022-10-11 江南大学 一种制备卵黄免疫球蛋白的方法
WO2022118114A1 (fr) * 2020-12-04 2022-06-09 3M Innovative Properties Company Unité de filtration et procédé de purification de biomatériau
CN114014926B (zh) * 2021-11-20 2023-08-18 江苏天美健大自然生物工程有限公司 一种简单快速从牛初乳中制备高纯度免疫球蛋白g1和g2的方法

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