DK153521B - PROCEDURE FOR THE PREPARATION OF PROTEIN CONCENTRATES CONTAINING MILK IMMUNOLOGICAL FACTORS - Google Patents

Description

DK 153521 B

The present invention relates to a process for the preparation of protein concentrates containing immunological factors from milk, especially active non-denatured immunoglobulins.

Immunological factors derived from milk in dietary products for newborns and breast-feeding children have already been proposed by incorporating these factors into the milk, the oral ingestion of these products will allow transport into the breast-mother's blood of these factors without at the same time seeing the attendant circumstances or the consequences of such a general passive immunization.

In the specification DE 1 810 438, for the purpose of immunization of calves, it is proposed to isolate the immuno-lactoglobulins from the milk from vaccinated cows by coagulation of milk, by extracting the

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toserum and selective precipitation of the lactoglobulins by e.g. ammonium sulphate followed by dialysis against clean water, filtration and drying. Examination of serous protection in mice in this connection has shown that parenteral injection of these immunizing principles produces a general passive protection against certain enteropathogenic bacteria and viruses if the microorganisms are introduced into the animals in the same way.

Also mentioned is Danish Patent Specification 87,610, which discloses a method of preparation for antigen protectors. Selected antigens are injected into the udder of a claw animal during the non-milking period of the animal and infusions continue during the milking period. Milk or colostrum is collected, foamed and casein removed by acid precipitation. Then, γ-globulin is precipitated with cold alcohol, dispersed in an aqueous saline solution, re-precipitated with alcohol and lyophilized.

U.S. Patent No. 3,911,108 discloses similar methods of preparation for antigens. These protective substances must be used on piglets.

The method uses a chemical antibacterial agent, namely β-propiolactone, to sterilize and separate antibody by precipitation with ammonium sulfate and subsequent dialysis.

None of these approaches are particularly suitable for industrial exploitation.

It has now been found a method to produce, on an industrial scale, protein concentrates containing immunoglobulins without denaturing them by technological processes. This product produces a local passive immunization in the intestine without resorption and without remarkable destruction of digestive tract activity.

The method of the invention is characterized in that it a) collects from milk-producing ruminants, especially cows that are hyper-immunized by vaccination, with successively administering antigens parenterally and locally. 8 to 1-2 weeks before calving and orally in the ca. 2 weeks before the calving and, optionally, a series of successive administrations of antigen from ca. 2 \ month before the supposed cessation of milk secretion with the switch between parenteral, local and oral administration. The above milk consists of colostrum from 1 and 2 hours after calving, transition milk from 3-8 days after calving and, optionally, milk from the last 60 days of lacta- 3

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tion period; b) separating the cream and impurities; c) coagulates the clarified and foamed milk; d) separates the casein, filters./ ultrafiltrates and sterilizes the protein from the whey by filtration; and (e) evaporate and dry the product under conditions such as not to denature the immunoglobulins and preserve sterility.

The accompanying drawing is a schematic representation of the various steps of the method.

It is preferred to treat a mixture of the above milk species to obtain an elevated antibody amount during the period as long as possible.

Hyperimmunization of the cow is performed by a combined vaccination. Eg. by intracisternal insertion into the mammary gland, by parenteral injection (subcutaneous, intravenous), by injection into the retromammary lymphatic system, by scarification, by oral administration, or by a combination of several methods.

It is preferred to use a combination of the methods, following a carefully laid immunization schedule to obtain a satisfactory antibody titer in each type of milk used. Thus, the vaccination for colostrum and the transitional milk is parenteral and intracisternal for 8-2 weeks before the calving and oral for the week before the calving.

For the milk from the end of the lactation, a vaccination change between parenteral, local and oral administration is used from 2.1 / 2 months before the expected cessation of milk secretion.

The vaccine used is advantageously a selected mixture of antigens and an adjuvant based on homologous blood serum from immunized cows, thus allowing a detoxification of the vaccine and the formation of immune complexes.

The cow serves as a production animal, the Ig compounds contained in the protein concentrate are, in principle, IgG compounds (especially IgG ·) different from the breast milk, which predominantly contains IgA as the Ig compound.

The content of Ig in the protein concentrate, of which the proteins make up 70 to 80% by weight, is 25 to 35% by weight. Certain advantages are related to the fact that the Ig compounds do not at the i

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4 initiation is separated from the other proteins in lactose serum, which are thus also present in the obtained concentrate. Thus, the selective precipitation steps are avoided, e.g. with the ammonium sulphate and dialysis.

Further, certain bacteriostatic or antivirus factors are found in the protein concentrate, e.g. the lactoferrin, or the enzymatic systems, such as lactoperoxidase, ribonuclease, which is present in the lactose compartment.

All kinds of antigens of virus or bacterial origin can be used and the antibodies obtained are dependent on the nature of the antigens administered to the cow. One can, for example. obtain protection against enteropathogenic bacteria and viruses responsible for gastroenteritis with concomitant vigorous diarrhea with dehydration, by incorporating protein concentrates containing the Ig compounds in all grants used for oral administration. This supplement may be any adjuvant or preferably a dietary product such as milk or milk powder for toddlers, a so-called "humanized" milk or adapted to breastfeeding children, milk specially adapted to a special group of newborns, such as milk for premature babies. or children with low birth weight, etc.

If you want to use e.g. a concentrate containing Ig anti E. coli in prophylactic dose with milk as a supplement, incorporate from 0.8 to 3 g of the concentrate in 100 g of the dry material and up to 6 g in the case of a therapeutic dose.

In order to carry out the procedure, it is necessary to make sure that the foaming steps and the clearing are carried out very carefully to avoid a subsequent clogging of the filters and ultrafilters.

The foaming is preferably carried out in two stages, first during cooling to remove most of the fats and impurities (e.g., blood often found in colostrum) and finally under heating to completely separate them. The coagulation can be carried out at the isoelectric pH of the caseir in the presence of cheese run with the addition of acid, e.g. citric acid.

It is preferred to carry out the coagulation by acid addition, e.g. hydrochloric acid to a pH of about 4.6 and heating to 40 ° C.

The separation of the casein results in a clearance of the serum which allows the separation of the precipitates.

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It is necessary to carry out the steps which necessitate a thermal treatment (evaporation and drying) under controlled conditions to avoid inactivation of the Ig linkers.

In order to reduce the losses, some of which are removed during the foaming of the cream and another with the casein when separated, it is recommended to extract the cream and buttermilk with water and treat the wash water containing the Ig compounds to recover them; In the following examples, the invention is further elucidated in that quantities and parts are given in units of weight unless otherwise stated.

Example 1 Cows of different breeds were hyperimmunized following the schedule given with a vaccine whose preparation is listed below and the milk was collected during the last two months of the lactation period and the first eight days after calving.

Preparation of vaccine:

The following enteropathogenic serum types of E. coli were used: 0 18: K 76 (B 20) 0 111: K 58 (B 4) 0 20: K 17 (L) 0 112: K 68 (B 11) 0 26: K 60 (B 6) 0 119: K 69 (B 14) 0 44: K 74 (L) 0 124: K 72 (B 17) 0 55: K 59 (B 5) 0 125: K 70 (B 15) 0 78 : K 80 (-) 0 126: K 71 (B 16) 0 86: K 61 (B 7) 0 127: K 63 (B 8) 0 128: K 68 (B 12)

The various strains produced gastroenteritis in hospitalized children. Serotyping was performed with multis and monovalent (Difco) antisera. The bacteria were grown on liquid minimum substrate containing 2% amino acids from casein (casamic acid Difco) and 2% glucose for 24 hours at + 37 ° C without shaking the flasks. To inactivate the bacteria, cultures were separated by centrifugation into settled cells and supernatant. The cells were resuspended in suspension and incubated at 37 ° C for 24 hours in the presence of 0.5% formaldehyde, while the supernatant liquid was inactivated as indicated but with 0.05% formaldehyde. After a new centrifugation and removal of the supernatant, the bacteria were placed in slurry in the initially inactivated supernatant. This procedure allows the supernatant fluid containing bacterial exotoxins to be retained for the vaccine.

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A vaccine with 1-2 x 10 bacteria / ml was obtained, and then the mixture was diluted with a solution of 2% Al (OH) g and homologous blood serum from immunized cows.

Method of immunization.

A. Immunization Scheme to Obtain Colostrum and Transition Milk Containing Ig Anti-E.coli Compounds:

Days of expected calving = X

Time of treatment Vaccine + possibly Adjuvant Method of Administration 7 X - 8 Weeks 5 ml Vaccine (5 x 10 Subcutaneous Injection Bacteria / ml) ^ + 5 ml Serum 7 X - 7 "5 ml Vaccine (5 x 10 Intravenous Injector Bacteria / ml + 5 ml 2% Α1 # (ΌΗ) 3 + 5 ml serum 7 X - 6 "10 ml vaccine (5 x 10 Subcutaneous injection bacteria / ml 7 X - 5 1/2 weeks 20 ml vaccine (5 x 10 injection in the re-bacteria / ml + tromammary) lymphocyte-10 ml 2% Al (OH), stem ό 7 X - 5 weeks 40 ml vaccine (5 x 10 Infusion of 4 x 15 bacteria / ml) ml in the udder with pat- + 20 ml serum tea needles Q ££ X - 4 1/2 weeks 24 ml vaccine (5 x 10 Subcutaneous injection bacteria / ml) + 8 ml serum * 7 X - 4 weeks 40 ml vaccine (5 x 10 Infusion of 4 x 15 bacteria / ml) ml in the udder with patina. + 20 ml serum tea needles

ISLAND

X - 3 "10 ml vaccine (5 x 10 Subcutaneous injection bacteria / ml) 8 X - 2 weeks 5 ml vaccine (5 x 10 Intravenous injection bacteria / ml), + 5 ml 2% Al (OH), X - 1 week 1 (50 ml xaccine · 3 (1 x 10y bacteria / ml) Oral administration during rumination X - 1/2 week 100 ml yaccine (1 x 10y bacteria / ml) Oral administration during rumination + These serum additions are only made when needed to it, or a cow being immunized for the first time.

++ This subcutaneous injection is repeated in the lymphatic system as follows: DK 153521 7 2 x 4 ml in the pre-scapular lymphatic systems 2 x 4 ml in the post-scapular lymphatic systems 2 x 4 ml in the lymphatic systems 2 x 4 ml in the poplitic lymphatic systems .

B. Immunization schedule to obtain the milk from the end of lactation (two last months of lactation) containing Ig anti-E.coli compounds.

This immunization is only performed on cows that have already been immunized during the colostral period and the transitional period.

Number of days for cessation of milk secretion = X Time of treatment Vaccine + exercised. adjuvant. Method of administration 7 X - 70 days 5 ml vaccine (5 x 10 Intravenous injection bacteria / ml) + 5 ml 2% Al (OH) - a X - 65 20 ml vaccine ^ 5 x 10 Injection into the retro-bacteria / mammary lymphatic system X - 60 "24 ml vaccine (5 x 10 ^ Subcutaneous injection ** bacteria / ml) (as under A) X - 55" 100 ml vaccine (1 x 10 ^ Oral administration under bacteria / ml) rumination 7 X - 50 "40 ml vaccine (5 x 10 Infusion in the udder as

bacteria / ml + 20 ml in A

serum 9 X - 40 100 ml vaccine (1 x 10 Oral administration under bacteria / ml) rumination

ISLAND

X - 30 "20 ml vaccine (5 x 10 Injection into the retro-bacteria / ml) mammary lymphatic system 7 X ~ 25" 40 ml vaccine (5 x 10 Infusion into the udder as

bacteria / ml) + 20 ml in A

"Serum g X - 10 100 ml vaccine (1 x 10 Administration under ruminant bacteria / ml).

Example 2 Milk from the first eight days after the calving was collected from vaccinated cows as described in Example 1 and treated as follows:

The foaming was performed in two steps: step A cold and step B hot. A. 1100 kg of milk was cold added to a foam centrifuge and piped to a sealed container of 1500 liters, after which it was pumped into a sludge centrifuge at elevated accelerations (about 11000 g) to eventually remove blood and impurities (dirt).

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B. The slurry entrif soaked cold milk was stored in a 2500 liter container and pumped through a tempered heating device of 40 ° C into the foam centrifuge, which had already been used, separating 110 kg of cream collected and 5 kg dirt, which one wasted. As a variant, a self-discharging sludge foam can be used for the cold and hot foaming step and a sludge centrifuge between the operations, thus reducing the duration of the foaming.

The frothed and de-skimmed hot milk (985 kg) was then placed in four square coagulation vessels on each 750 liters equipped with a pipework to coagulate.

A solution of hydrochloric acid of 1 N is added at 37 ° C until the pH stabilizes around 4.6 and the temperature is maintained for 20 minutes with stirring. Then cool to approx. 15 ° C.

The next step is the separation of the casein. It consists of two centrifuges one after the other after the foaming of milk, coagulation and cutting. The first centrifugation separates most of the precipitated casein by means of a clearing autoclave trifuge, separating 150 kg of casein and the serum into a closed container. The second centrifugation removes all traces of particles that may interfere with subsequent filtration and ultrafiltration, e.g. by means of the sludge device used in Operation A for foaming. 854.7 kg of clarified serum is obtained, which is pumped into a 2500 liter container.

Thereafter, filtration is continued, followed by ultrafiltration of the serum. The filtration must be very careful to facilitate ultrafiltration, separating the very fine particles of casein. The "Seitz Supra" 100 filter is used in 20 x 20 cm pieces. The filtrate is stored in a 3000 liter reservoir and then pumped to the ultrafiltrate. The ultrafiltration takes place in two or three stages with recycling of the retained material (for convenience in the following called the retentate) to the reservoir. Two centrifuge pumps in series ensure flow against an input pressure in the ultrafilter's input section of approx. 7 bar, keeping the initial pressure of approx. 4.5 bar. To avoid reheating the retentate at the energy coming from the pumps, this is cooled to 10-12 ° C.

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The first step or ultrafiltration itself allows separation of the high molecular weight solutes, the proteins retained on the membrane, which are present in the retentate, while a portion of the lactose, vitamins, mineral salts and water are transferred to the permeate. If using a DDS model with membrane 800 (surface-2 membrane = 7 m), the retentate / permeate ratio will be approx. 1 to 3.1, thus obtaining 580 kg of permeate I and 274.7 kg of retentate with a flow rate of 14.17 kg permeate / m hour.

The second step is N a diafiltration during which 825 kg of water is continuously added to the retentate and the solution is circulated under the same conditions as found in the first step. The diafiltration is stopped when the electrical conductivity of the permeate achieves the desired value, the ratio of retentate / added water will be approx. In this way, 825 kg of per-meat II and 274.7 kg of retentate are produced with an average flow of 11.77 kg per-2 meat / m hour. The third step is a concentration where, if desired, the retentate is recirculated on the membrane until a dry matter content of approx. 10%, removing 82.5 kg of permeate III. Thus, 192.2 kg of preconcentrated solution is obtained. Alternatively, you can omit this third step and go straight to sterile filtration.

Sterile filtration is an important step in the whole process because heat treatments are not useful for sterilization as they will lead to denaturation of the Ig compounds. The sterile filtration removes microorganisms from the retentate that may still be found there (the majority of these have already been removed during the separation of the cream, casein and impurities). To avoid clogging of the sterile filters, removal of impurities by centrifugation and a third filtration of the retentate is necessary. After removal of impurities, the retentate is stored in the reservoir and passed along a filtration line which includes a pre-filtration of three filters "Seitz Supra" 100, ΕΚ, ECS placed in series, then sterile filtration on the "Seitz Supra" 20 and "Millipore" filters. HA 0.45µ arranged in series and preferably sterilized with overheated water. The sterilized retentate is stored in a 500 liter sterile reservoir.

All other operations are done under sterile conditions. By

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With the help of compressed nitrogen and sterile filter, e.g. transfer to the subsequent evaporation.

The evaporation is carried out with a film evaporator (with a stream falling 2 over a surface of 1 m) at a heating temperature of 75 ° C, the temperature of the product being about 30 ° C to a dry matter content of at least 20%. To avoid any infection, the transfer is performed with a peristaltic pump from the retentate reservoir to the concentrate reservoir, which is connected in a ring comprising the evaporator, the concentration taking place in a closed circuit. This operation does not affect the activity of the Ig compounds.

Drying of the concentrate (96 kg) is then continued by ordinary methods, e.g. by freezing followed by lyophilization. The freezing can be carried out on plates of -40 ° C. The Ig compounds can be stored at -30 ° C without losing activity for approx. 45 days. The product at -30 ° C is lyophilized on plates in an oven at 0.5 torr at a condensation temperature of -50 ° C and a heating temperature of 30-35 ° C. The drying method does not affect the activity of the Ig compounds. In this way, 19.1 kg of a dry product containing 25-35% Ig compounds are obtained which are sterilized conditioned.

Example 3

Proceed as in Example 1, treating the milk from the first eight days after the calving and the milk collected during the last four weeks of the lactation period to obtain a protein concentrate with the average composition:

Proteins 75-5% 40 -5% immunoglubolines 15-5% α-lactalbumin 35-5 3-lactoglobulin 5 -2% Serum albumin 5 -2% Other minor proteins Residual moisture 4-0.5% Lactose 10-2% Mineral salts 5 -2% nitrogen compounds, + non-proteins. 5-2% 11

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Example 4

Proceed as described in Example 2, but with the difference that the cream and casein are extracted with water to recover some of the lost Ig compounds as the wash water is recycled on the fabrication line.

The cold foaming (stage A) is carried out in a line parallel to the water extraction of the protein fraction containing the Ig compounds removed with the cream. The cream resulting from the first spin (115 kg) is stored in a 1000 liter reservoir with stirring and mixed with 150 kg of lukewarm water at 40 ° C, then mixed for 30 minutes and centrifuged in a dairy centrifuge.

Thus 115 kg of cream and 150 kg of solution are separated, which are combined with the foamed and clarified milk. In this way, 130 kg of liquid is obtained, which is led to hot foaming (stage B) and coagulation.

The separation of the casein results from 1145 kg of skimmed milk added to rennet and acidified, 991 kg of serum and 154 kg of casein. A parallel line allows to extract the protein fraction containing the Ig compounds coagulated in water. The coagulate is stored at the exit of the clearing autoclave centrifuge in a reservoir equipped with a stirrer and stirred with 300 kg of lukewarm water at 40 ° C for 30 minutes, after which it is fed to a colloid mill. The suspension of the ground clot is then separated into a clearing autoslice centrifuge. 154 kg of washed casein and 300 kg of washing liquid are extracted, which is combined with the serum from the other operations. 1291 kg of serum is filtered and ultrafiltered to give 2010 kg of permeate (I, II and III) and 216 kg of preconcentrate. Evaporation gives 108 kg of concentrate, which gives 21.6 kg of dry product on drying. Thus, you get approx. 14% Ig compounds more than by the method of Example 2.

Example 5 1 kg of powder prepared as in Example 2 or 4 or 2 kg of powder prepared as in Example 3, respectively, are mixed with 100 kg of milk powder in a uniform manner and this is sterilized to form a product containing one prophylactic dose in one nutrient dose corresponding to 140 cal / kg / day corresponding to 250 mg concentrate Ig / kg / day prepared as in Examples 2 or 4, respectively to 500 mg concentrate Ig / kg / day prepared as in Example 3.

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Example 6

Various experiments in vitro and in vivo have been carried out with the protein concentrate prepared as in Examples 2 or 4 and hereinafter referred to as "concentrate Ig" to show: that the Ig milk compounds have an antibody specificity normally found in the human Ig compounds. , and which is retained during the manufacturing process that the specific Ig milk compounds exhibit a protective activity, interfering with the pathogenic mechanisms of entero-pathogenic microorganisms.

Passive hemagglutination.

Red blood cells from sheep were sensitized with a urea extract of specific serotype E. coli. This extract was prepared as described by Brodhage (Brodhage H., (1961) J. Hyg., 1961, 148, 94). The bacteria were grown on nutrient agar ("Difco") at 37 ° C for 30 hours in a Roux flask. The culture was harvested with 10 ml of a saline solution buffered with phosphate (8.8 g NaCl + 1.86 g · 2H 2 O + 0.43 g KH 2 PO 4/1000 ml H 2 O) until pH 7.2. After adding 10 g of urea ("Merk"), the slurry was allowed to stir for 90 hours at 37 ° C. After centrifugation, the supernatant was completely dialyzed against a saline solution buffered with phosphate to pH 7.2, diluted to a final volume of 50 ml with a saline solution buffered with phosphate to pH 7.2, then frozen in small portions at -20 ° C. Sensitization of red blood cells from sheep with this antigen was performed by a combination of the methods described by Avrameas et coll. (Avrameas S., Taudou B., Chuilon S., Immunochemistry, 1969, j5, 67) and Otto et al. (Otto H., Takamiya H., Vogt A., J. Immul. Methods, 1973, 3 ^ 1937). pre-treated with glutaraldehyde (final red blood cell concentration from sheep 5% and 0.5% glutaraldehyde), then washed and slurried with 20% of a saline solution buffered with phosphate to pH 7.2, and mixing the same extract volume with urea. 16 hours incubation at 20 ° C under 1 ° After stirring, the sensitized red sheep blood cells were washed several times and slurried up to 1% for immediate use. The red sheep blood cells can be stored in a 10% suspension at 4 ° C for several weeks.

Prior to filtration, each sample to be examined should be absorbed on red sheep blood cells pre-treated with glutaraldehyde (0.1 ml red sheep blood cells precipitated per 1 ml Ig concentrate, 37 ° C, 60 ml).

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Nutter).

The titrations were performed with the microtiter system (Sever JL, J. Immul., 1962, 8j3, 320, and Conrath TB, Handbook of Microtiter Procedures, 1972), using polystyrene plates with hollow-out v-shaped plates. in normal rabbit serum diluted to 1/200, and 25µ liters of sensitized red sheep blood cells of 1% were added to each dilution. A series of non-sensitized red sheep blood cells were prepared as nonspecific agglutination control. The results were read after 2 hours, indicating the hemagglutination titer at the last dilution that led to a clear positive reaction.

The results obtained for 5 serotypes of E. coli are given in the table below.

Serotype E.coli_Agglutination titers 0 55 1/256 0 111 1/64 0 119 1/128 0 127 1/128 0 128 1/64 control -

Bacteriostatic activity in vitro.

The bacteriostatic effect on the growth of various serotype E.coli was investigated by the addition of Ig concentrate during culture. The microtiter system adapted for cultivation was used, which allowed the use of minimal sample quantities and at the same time investigated a maximum number of samples.

Each culture had a final volume of 0.25 ml partly on a substrate containing at least 1% glucose and partly on a rich culture substrate. For each cultivation time studied, two samplings were performed to reduce the error due to variations in volume. The assessment was carried out by double counting 10µ liters on nutrient agar plates. The bacterial inoculum consisted of a dilution of 3 · 16 hours of culture from an environment containing at least 1 to 2 x 10 bacteria / ml. The incubation was performed in a humid atmosphere at 37 ° C.

Ig concentrates at final values of µg / ml, 10μg / ml, 3 100µg / ml and 1 mg / ml were added to the culture substrate prior to inoculation with 2 x 10 5 E. coli / ml. A pure growth inhibition of E. coli 0 111: B4 was observed over a 4-hour incubation with an Ig concentration of 10μg / ml.

By comparison with the control which consisted of the culture substrate

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14 alone, a better growth was found in the presence of 1 mg / ml of skimmed milk protein from non-immunized cows.

Clearance of E. coli 0 111: B4 in mice.

For each experiment, 8 mice (white Swiss male mice of 20 to 24 g) were used, 2 as controls and 6 to 3 experiments each performed twice. All mice received a subcutaneous injection of 0.1 ml of heparin (500 UI / ml). A 16 hour culture broth of E. coli 0 111: B4 was diluted to 1/10 with a sterile saline solution and then diluted to 1/100 by the addition of fetal calf serum ("Difco") diluted 1/10 as controls and containing three selected cones. concentrations of Ig concentrate. 0.2 ml of the obtained solution was injected intravenously into the caudal vein, each mouse (2 mice being

C

controls and 2 x 3 as experiments) were given approx. 2 x 10 bacteria, the

Q

original bacterial slurry contained 10 bacteria / ml. Immediately after injection (at time t0) and after 20, 40 and 60 minutes, a 50 µl blood sample was taken from the ophthalmic vein plexus using calibrated heparinized capillary tubes and the samples were immediately transferred to 5 ml sterile saline solution (blood 2 -3 -4 dilution 10) and then diluted to 10 and 10 with brine. The count was performed on agar plates ("Difco") with 1 ml of each dilution and the phagocytosis index K determined

Biozzi et coll. (Biozzi G., Stiffel C., Halpern B.N., Le Minor L.,

Mauton D., J. Immul., 1961, 87 (296): log C-, - log C9 K = ---— T2 - ^ C · ^ and C2 corresponding to counts at time and T2.

Thus, a normal decrease in the number of bacteria was observed by elimination through the reticulo-endothelial system in the presence of fetal calf serum, while the addition of 10g of Ig concentrate in the injection solution caused a 99.6% disappearance of the bacteria from the blood stream over 20 minutes.

The results are calculated in the table below:

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The time Bacteria in% of the original number of infusions (minutes after the 0 after intravenous injection of 2 x 10 ° injection E. coli 0 111: B4 of E. coli) _

Control 1:10 Samples Ig concentrate fetal calf 1000 µg 100 µg 10 µg serum 0 100 100 100 100 20 27.8 0.14 0.22 0.4 40 ------------- 15.8 ----------- 0.02 ...... 0.02 · - "------- 0.12 '60 10.0 0.02 0, 02 0.06

The specificity of opsonization was checked by complete absorption of the Ig concentrate with serotype 0 111: B4 E. coli and it was found that this absorption almost destroyed the entire increase in activity of the phagocytosis even with an amount of 1 mg Ig concentrate.

The table below provides phagocytosis K values obtained over 20 minutes.

Control 1000 g Ig- 1000 g Ig- 1:10 fetal concentrate calf serum unabsorbed absorbed K 0.015 0.128 0.039

Experiments with protection in mice.

Logarithmic dilutions (log ^g) of a culture of E. coli 0111: B4 were prepared in food broth ("Difco") containing 9 -4 -4 -5 -6 containing 10 bacteria / ml for dilutions of 10, 10, 10 and 5% in mucin of 5% (granular mucin, type 1701-W to potentiate the virulence of bacteria from Wilson Laboratories, Chicago, Illinois).

3 ml of each dilution was then mixed with 0.6 ml of test solution, respectively with saline solution, Ig concentrate, proteins from common whey (non-immunized cows). Using 5 mice for each dilution, 0.6 ml of the obtained mixture was injected intraperitoneally into each mouse. The test results in the form of dead mice per Five treated mice after 48 hours are listed in the table below.

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Experimental Number of dead mice / 5 treated "inus.

Concentration of bacteria in the treatment 5 x 104 5 x 103 5 x 102 5 x 101

Saline solution 5555 1 ° μ *

Ig Concentrate 5553 25µ *

Ig Concentrate 5400 100µ

Ig concentrate 0000 10µ proteins from common whey (non-immunized cows) 5555 25µ proteins from common whey 5 5 5 5 100µ proteins from common whey 5 5 5 5 calculated as total proteins containing 35-45% Ig compounds from milk.

It was observed that 100 µg Ig concentrate provided complete protection against all bacterial sample concentrations, while 100 ng of whey proteins isolated from non-immunized cows provided no protection.

Example 7 I. A first series of clinical trials showed that Ig compounds from milk resisted proteolytic degradation to inactive parts of the intestinal tract.

11 children (9 boys, 2 girls) hospitalized for various reasons, but not suffering from gastroenteritis, and whose age was from 2 weeks to 1 year, fed with milk containing an Ig concentrate prepared as in Example 2 or 4 in caloric doses corresponding to 2 g / kg body weight distributed uniformly over 24 hours. The first and last meals in which Ig concentrate was incorporated were labeled with 0.2 g of carmin red. At least one stool sample was collected prior to the administration of Ig and systematically all subsequent stools over 72 hours, the stool being stored at -20 ° C.

Extracts of the stools were prepared by adding 2 parts of stool to 1 part saline solution buffered with phosphate to C tr- λλλ f - »- 17

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pH 7.2 and dispersed the stools at 0 ° C with a glass spatula. The suspension was then stirred for 20 minutes with a mini stirrer (400 rpm) at room temperature. The suspension was then cooled rapidly to 0 ° C and centrifuged at this temperature at 3500 x g for 15 minutes. Finally, the supernatant was carefully removed.

The presence of active Ig compounds in the stool extract was confirmed by double immunodiffusion (Ouchterlony's trial) and by immunoelectrophoresis. Antisera used for these experiments was prepared by subcutaneous and intravenous injection repeated several times by 1 mg of milk antibody or 0.1 mg of Ig G1 on rabbits. For Ouchter-lony's experiment, glass plates (94 x 84 mm) were covered with a layer of a 1% agar gel in a saline solution buffered with phosphate to pH 7.2, in which gel was cut with a space of 4 mm at 9.5 mm with the punching machine ("LKB-Produkteur AB"), using an anti-protein antiserum from colostrum whey from cows and a monovalent antiserum specific anti-Ig from cow's milk.

For the immunoelectrophoresis, the glass plates (100 x 85 mm) were covered with 12 ml of 1% agar gel in 0.05Ή barbiturate buffered to pH 8.3 (10.3 g sodium barbiturate + 0.5 g citric acid + 0.5 g oxalic acid in 1 liter water) and the electrophoretic separation was performed at room temperature over 90 minutes at 4 volts / cm. After diffusion of antisera (24 hours), the plate was washed with a saline solution, dried and developed with azocarmine B.

The presence of milk Ig compounds in the stools was observed, although there were significant differences in when they appeared and in the duration of the secretion of Ig, as well as a good agreement between the appearance of the red carmine color and the Ig compound and disappeared.

To confirm the activity of the Ig milk compounds, a test was performed in vivo on seroprotection in mice (as described in Example 6), using 6 stool extracts in each row, the results of which are given in the following table: 18

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Extract of Intensity of Ig Number of dead mice / faeces in faeces-treated mice funnel by im- Number of bacteria used in the microelectrophoresis in the treatment study. , ~ ·, 5x10 5x10 5x10 ^ 5xlOx

Children M.D. I 5 5 5 5 (2 weeks) II 5 5 5 5 IV + + + + 2 1 0 0 V + + + + 0 0 0 0 VI + + + 1 1 0 0 IX -; - 5 5 3 4

Children S.G. I -: - 5 5 5 5 (1 month) III 4 5 5 5 IV + + + + 0 0 0 0 VI + + + + 0 0 0 0 X + + 3 0 10 XI + 5 2 0 0

There was a clear correlation between the positive results of immunoelectrophoresis and the protective ability of the material contained in the faeces extracts. Specifically, the concentration of Ig milk compounds from extracts V (child M.D.) and IV and VI (child S G.) was determined corresponding to at least 1 mg of Ig concentrate.

II. In a second series of clinical trials, the therapeutic and prophylactic properties of Ig concentrates prepared as described in Examples 2 or 4 were investigated.

Therapeutic properties.

This study was conducted with children up to 5 months suffering from benign to severe gastroenteritis induced by E.coli. In various series of experiments, a total of 152 patients were administered 2 g of concentrate Ig / kg / day for 5 days and 1 g / kg / day over 10 days for their food. Patients received no drugs such as sulfamides or antibiotics, either orally or parenterally.

Culture was performed from the faeces the day after the administration, and the last between 36 and 48 hours after the last Ig concentrate administration. 43 Patients were treated similarly, but replacing the Ig concentrate with whey proteins originating from non-immunized cows.

Negative faecal cultures were obtained for 90 children (57.7%) during the treatment. Only 14 children (27.7%) showed

So be it

DK 153521 B

19 tan the disappearance of E.coli from the cultivation of the face tests. It should be noted that natural proteins derived from whey from non-immunized cows contain a low amount of Ig anti-E.coli. It was also observed that the appearance of successful outcome of the treatment was seen more often in cases where high doses were used over a very short period of time.

The consistency of faeces was examined in each case. It was found that diarrhea disappeared and a marked clinical improvement in a large number of cases, even if stool specimen culture remained positive. In the case where fecal culture culture became negative, the stool consistency improved faster if the Ig concentrate was administered in a milk powder poor in lactose.

Prophylactic properties.

The premature babies form a group that is extremely sensitive to gastroenteritis by E. coli, and therefore this group was selected for a clinical prophylaxis trial.

The study was conducted over 6 months in 2 living rooms with 8 incubators in each living room. The trials and controls were distributed in each living room so that all patients were subjected to the same conditions, with 4 incubators serving as trials and 4 as controls. Each case lasted an average of 41 days, with one child removed after healing and replaced by another dependent on affiliation (trial or control). 70 cases were followed, 36 as control and 34 as experimental group.

The experimental group received Ig concentrate at each feeding with milk distributed over 24 hours at 0.25 g / kg / day. Stool samples were examined from the beginning of the experiment and stool samples were examined by cultivation every five days. Of a total of 666 faeces grown, 339 controls and 327 were test samples. Among the latter, 33 showed the presence of E.coli (10.1%), while 96 of 339 control cultures were positive (28.3%). Examining only E. coli 0 119: B14 serotype, it is often associated with severe gastroenteritis, and the rate of positive faecal culture was 5.5% in the experimental group versus 23.3% in the control group.

It can be concluded that the incorporation of protein concentrates containing milk-specific anti-E.coli Ig.to the premature milk significantly decreases the rate of infection with E.coli in this particularly sensitive group of newborns.

Claims (9)

A process for producing protein concentrates containing immunological factors from milk, especially active non-denatured immunoglobulins, characterized in that: locally approx. 8 to 1-2 weeks before calving and orally in the ca. 2 weeks before the calving and, optionally, a series of successive administrations of antigen from ca. 2 \ way before the assumed cessation of milk secretion with the switch between parenteral, local and oral administration; the above milk consists of colostrum from 1 and 2 hours after the calving, transition milk from 3-8 days after the calving and, optionally, milk from the last 60 days of the lactation period; b) separating the cream and impurities; c) coagulates the clarified and foamed milk; d) separating the casein, filtering, ultrafiltrating and sterilizing the protein from the whey by filtration; and e) evaporating and drying the product under conditions not denaturing the immunoglobulins and preserving sterility. Method according to claim 3, characterized in that the vaccine is based on Escherichia coli antigens which cause gastroenteritis in neonates. Process according to claim 1, characterized in that the coagulation is carried out by addition of hydrochloric acid until the pH is approx. 4.6 and heating up to 40 ° C. Method according to claim 1, characterized in that, in order to avoid the following clogging of filters and ultrafilters, the foaming in two stages is first carried out cold and then hot, and the foaming and separation of the casein is followed by a forced clearance. Process according to claim 1, characterized in that the ultrafiltration of the whey is carried out with washing of the retentate and recyclization of the washed retentate, so that its content of proteins is increased. Process according to claim 1, characterized in that the preconcentrated whey is subjected to a pre-filtration followed by sterile filtration. DK 153521 B Process according to claim 1, characterized in that the evaporation of the preconcentrated and sterilized whey takes place in a closed system under sterile conditions. Process according to claim 1, characterized in that the concentrated product is dried by freezing followed by lyophilization and sterile conditioning. Method according to claim 1, characterized in that to recover the immunoglobulins removed during steps b) and d), the cream and the thick milk are extracted with water and the washing water is combined with the foamed milk and the whey respectively.