GB2179947A - Process for the extraction of proteins from milk - Google Patents

Abstract

A process for the extraction of proteins, preferably lacto-transferrins or IMMUNOGLOBULINS, from milk from which the casein and the fatty substances have been substantially removed, comprises adsorbing the proteins on an ion exchanger followed by elution whereby the desired protein fraction is obtained, the adsorption and the elution being carried out at substantially the same pH, preferably 5 to 8.5, especially 7 to 8. The casein-free milk is preferably concentrated about five times, by ultrafiltration, before adsorption.

Description

SPECIFICATION Process for the extraction of proteins from milk The subject of the present application is a process for the extraction of proteins from milk and in particular those proteins capable of fixing iron, the use of such a process in the preparation in particular of lactotransferrins, the products obtained by the process and pharmaceutical compositions containing them.

Processes for the preparation of proteins, particularly lactotransferrins and/or immunoglobulins have already been described. There can be cited notably French patent No. 2,505,615 and the article by Cheron [C.R. Acad. Sc. Paris t 284 (14 February 1977)].

These processes do not, however, allow lactotransferrins to be obtained of a quality, with a yield, and from a material suitable for industrial use.

We have now found that lactoproteins, and in particular lactotransferrin, may be obtained in a single, adsorption-elution stage on an ion exchanger, under non-denaturing and particularly mild conditions, using a light industrial material, and in a reduced time, if the adsorption and elution are carried out at the same pH.

Thus according to the present invention we provide a process for the extraction of proteins from milk from which the casein and the fatty substances have been substantially removed, which comprises adsorbing the proteins on an ion exchanger followed by elution whereby the desired protein fraction the adsorption and the elution being carried out at substantially the same pH. The process of the present invention is of particular use for the extraction of proteins able to fix iron.

The ion exchanger used can be an anion exchanger, but is, for preference, a cation exchanger.

In order to obtain a more rapid and easier elution so as to avoid denaturing the proteins, it is preferred to use a weak cationic resin, for example, a carboxymethyl resin such as those marketed under the trade names CM Trisacryl (IBF) and CM Sepharose (Pharmacia).

When a cationic resin is used, the adsorption and elution are carried out at a pH lower than the isoelectric point of the lactotransferrin, preferably at a pH of from 5 to 8.5, more preferably at from pH 6.5 to 8.3, and particularly at a pH of from 7 to 8.

The starting material is preferably a raw milk from which there has previously been extracted the greater part of the casein and the fatty materials. For example, a mild or acid lactoserum is used. The milk should not have been submitted to operations which destroy the proteins or degrade them, such as pasteurization under violent conditions, e.g. a few minutes at 90"C, or skimming. The raw milk can, for example, be decaseinated by precipitation e.g. with hydrochloric acid.

The separation of the curds and the lactoserum may be carried out according to the techniques usually employed for liquid-solid separations, such as, for example, decanting, centrifuging, or, preferably, filtering.

In the case of filtering, the membrane chosen should preferably be one which does not fix the proteins by, e.g. adsorption; for example, a membrane of cellulose nature is used.

In a preferred embodiment, a concentrated lactoserum is used in the adsorption stage. The lactoserum can be concentrated according to the usual methods such as e.g. by evaporation, for example from 2 to 20 times, and preferably about 5 times. The concentrated lactoserum is preferably desalinated, so as to diminish its ionic strength.

These latter preferred steps of concentration and adjustment of ionic strength may advantageously be carried out in a single stage by ultrafiltration. The type of ultrafilter must be chosen so as not to adsorb the lactoproteins excessively, and to retain them. The threshold of selectivity will be chosen, for example, between 10,000 and 70,000, and preferably between 25,000 and 50,000. The ultrafilter may be in plane or tubular form, or in the form of hollow or spiral fibres.

As ultrafilters which do not fix or only slightly fix lactoproteins, particularly lactotransferrin, there can be mentioned especially those of the polysulphonic type marketed under the name Millipore, notably those of type PSED 0HV 10.

The desalination and the concentration may thus be carried out by the same means, with the same apparatus; in this case, the desalination is preferably carried out by diafiltration (ultrafiltration under slight pressure) with 1/2 to 2 volumes of distilled water, for example.

The adsorption of the lactoproteins and their elution is generally carried out using the same concentration of buffer product in order to maintain the chosen pH, elution being effected by modifying the ionic strength, e.g by means of sodium chloride. The different ionic strengths are chosen so as to enable separation of the desired protein fraction, for example by eluting first the undesirable proteins, and only then the desired proteins. For example, the elution of proteins capable of fixing iron, notably the lactotransferrins, is advantageously preceded by from 1 to 5 elutions of lower ionic strength, and preferably from 1 to 3 elutions. The elution buffer is preferably anionic, such as a phosphate buffer.

The desired eluate is advantageously desalinated in order to eliminate the small molecules of molecular weight less than 1,000. Desalination may be carried out in the usual way, for example by filtering with a Millipore ultrafilter or by chromatography using gel permeation, G25 for example.

The process according to the present invention is particularly efficient since it enables pure lactotransferrin to be prepared from lactoserum by ultrafiltering and separating on a cation exchanger in 9 hours, whereas a process such as that of Cheron mentioned above requires 24 hours, i.e. about three times longer.

The substantially pure lactoproteins, in solution in the eluate, can with advantage be dried, particularly by lyophilization.

The iron-fixing proteins may advantageously be submitted to the action of a complexing agent, preferably a chelating agent, having a greater affinity than they have for iron, so as to increase their capacity for fixing iron. An EDTA phosphate buffer is used, for example.

The complexing agent can be eliminated by desalination according to the techniques already indicated.

The present application also has as its subject protein fractions obtained by the process described above, particularly lactotransferrins and immunoglobulins. The process described above is used with particular advantage for their preparation.

The therapeutic use of lactoproteins is well known, in particular, the bacteriostatic activity oflactotransferrin. For this reason, the present application has also as its subject pharmaceutical compositions containing the products obtained by the above described process. In this regard, it should be noted that lactotransferrin obtained by the above described process retains its bacter- iostatic properties.

As medicaments, the products obtained by the above process can be incorporated in pharrna- ceutical compositions intended for the digestive or the parenteral route.

The pharmaceutical compositions can be, for example, solid or liquid and may be presented in the pharmaceutical forms currently used in human medicine, such as, for example, plain or sug)ar- coated tablets, capsules, granules, suppositories, collyria or nasal solutions. They may be pre- pared-by the usual methods. The active principle or principles can be incorporated with the excipients usually employed in these pharmaceutical compositions, such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, fatty substances of animal or vegetable origin, paraffin derivatives, glycols, various wetting, dispersing or emulsifying agents, and preservatives.

The examples which follow illustrate the present invention, without however limiting it.

Example 1: Extraction of lactotransferrin Under agitation, 15.8 ml of 37-39% hydrochloric acid is added to 3.5 1 of raw milk, which is subsequently allowed to precipitate at 40"C for 1 hours, then filtered on a cellulose membrane.

The 2100 ml of lactoserum thus obtained is submitted to ultrafiltration on a Millipore membrane of polysulphonic type with a separation threshold of 25000 (reference PSED 0HV 10), by diafiltration with the same volume of distilled water, and is then concentrated to 420 ml.

The pH of the concentrated solution is adjusted to 7.0 with sodium hydroxide and the ionic strength is adjusted to 0.15 M with sodium chloride (measuring with a conductance meter). 400 ml of solution is then passed at a rate of 3 ml/min through a column of 25 mm internal diameter filled with 100 ml of CM Sepharose CL 68 (Pharmacia) previously balanced in a 0.15 M, pH 7.0 phosphate buffer. The column is eluted at a rate of 3 ml/min with a 0.1 M pH 7.0 phosphate buffer of which the ionic strength is adjusted by means of sodium chloride according to the following gradient: 270 ml at 0.15 M 145 ml at 0.25 M 145 ml at 0.30 M.

The lactotransferrin s eluted with 430 ml at 0.4M. Desalination is carried out on Pharmacia gel G 25, the solution obtained is lyophilized, and 84 mg of lactotransferrin are obtained.

Example 2: Extraction of lactotransferrin Decaseination and ultrafiltration are carried out as indicated in Example 1, starting with 1.5 1 of raw milk.

150 ml of solution is passed at a rate of 1 ml/min through the column. One elution is carried out with 150 ml of 0.15 M pH 7.0 phosphate buffer with an ionic strength of 0.25 M, then the lactotransferrin is eluted with 80 ml of pH 7.0 buffer with an ionic strength of 0.43 M.

After desalination and lyophilization, 40 mg of lactotransferrin are obtained.

Example 3: Extraction of lactotransferrin The lactoserum is prepared and deposited on a weak cationic exchanger as indicated in Example 1.

The column and the exchanger are then washed with 200 ml of 0.1 M pH 7.0 phosphate buffer, of which the ionic strength is adjusted to 0.15 M with sodium chloride. The lactotransferrin is then eluted with 100 ml of the same buffer, of which the ionic strength is adjusted to 1 M with sodium chloride. The desalination is carried out in the same way as previously (filtering on Pharmacia gel G25). After lyophilization, the yield of the operation is about 90 mg of lactotransferrin for 3.5 1 of milk.

Example 4: The lactoserum is prepared as in the previous examples. Its pH is adjusted to 7.0 with sodium hydroxide. AZARI and BAUGH solution is then added in an amount of 1 ml per litre of lactoserum.

Saturation of the lactotransferrin in ferric ions is allowed to take place at ambient temperature for 2 hours. After this, the solution is centrifuged and then ultrafiltered as in the previous examples.

400 ml of the material retained on the filter is combined with 100 ml of weak cation exchanger; after washing with 200 ml of 0.15 M, pH 7.0 phosphate buffer, the saturated lactotransferrin is eluted with 100 ml of the same buffer, of which the ionic strength is adjusted to 1 M with sodium chloride.

The preparation is desalinated by filtering on gel (G25) and then lyophilized.

The saturated and desalinated lactotransferrin is dissolved in a 0.05 M pH 4.5 phosphate/citrate buffer in an amount of 1 mg per ml of buffer.

The desaturation is carried out for 12 hours at +4"C. The desaturation solution of lactotransferrin is then desalinated by filtering on G625 gel, then lyophilized.

Composition of the AZARI and BAUGH solution.

Prepare a 0.1 M solution in sodium citrate and 0.1 M in sodium bicarbonate.

Add to 100 ml of this preparation 232 mg of ferric chloride-6H20.

Example 5: Some lactoserum is prepared and concentrated as in the previous examples. The pH of the concentrated solution is adjusted to 7.0 with sodium hydroxide.

10 litres of the preparation is passed at a rate of 6 litres per hour through a column 10 cm in diameter filled with 600 ml of CM Sepharose Fast-flow (Pharmacia) previously balanced in a 0. 15 M pH 7.0 phosphate buffer.

When this is done, the column is washed with a litre of 0.15 M pH 7.0 phosphate buffer, then with a litre of the same buffer of which the ionic strength is adjusted to 0.3 M with sodium chloride.

The lactotransferrin is then eluted with 600 ml of the same buffer, of which the ionic strength is adjusted this time to 1 M.

Desalination is carried out by filtering on G25 gel, then the preparation is lyophilized.

About 30 mg of lactotransferrin are obtained per litre of substrate.

Example 6: Pharmaceutical composition Nasal drops with the following composition have been prepared: - lactotransferrin 5 mg - stabilizer 250 mg - distilled water q.s. for 5 ml.

Claims (18)

1. A process for the extraction of proteins from milk from which the casein and the fatty substances have been substantially removed, which comprises adsorbing the proteins on an ion exchanger followed by elution whereby the desired protein fraction is obtained, the adsorption and the elution being carried out at substantially the same pH.

2. A process according to claim 1 for the extraction of proteins able to fix iron.

3. A process according to claim 2 wherein the elution of the proteins able to fix iron is preceded by 1 to 5 elutions of lower ionic strength.

4. A process according to claim 2 or claim 3 wherein, after elution the proteins able to fix iron are submitted to the action of a complexing agent.

5. A process according to any preceding claim wherein the ion exchanger is a cation exchanger.

6. A process according to claim 5 wherein the exchanger is a weak cationic resin.

7. A process according to claim 5 or claim 6 wherein adsorption and elution are carried out at a pH of from 5 to 8.5.

8. A process according to claim 7 wherein adsoption and elution are carried out at a pH of from 7 to 8.

9. A process according to any preceding claim wherein the milk from which the casein and the fatty substances have been substantially removed is concentrated about 5 times before adsorption.

10. A process according to claim 9 wherein concentration is carried out by ultrafiltration.

11. A process according to claim 10 wherein the cut-off threshold of the ultrafilter is between 25,000 and 50,000.

12. A process according to claim 10 or claim 11 wherein the ultrafilter has a polysulphonic nature.

13. A process for the extraction of proteins from milk according to claim 1 substantially as herein described.

14. A process for the extraction of proteins from milk substantially as herein described in any one of Examples 1 to 5.

15. Products obtained by a process according to any one of claims 1 to 14.

16. Lactotransferrins whenever obtained by a process according to any one of claims 1 to 14.

17. Immunoglobulins whenever obtained- by a process according to any one of claims 1 to 14.

18. Pharmaceutical compositions comprising at least one product as claimed in any one of claims 15 to 17.