CH668428A5 - PROCESS FOR THE EXTRACTION OF MILK PROTEINS, PRODUCTS, APPLICATION OF THE PROCESS AND PHARMACEUTICAL COMPOSITIONS. - Google Patents
PROCESS FOR THE EXTRACTION OF MILK PROTEINS, PRODUCTS, APPLICATION OF THE PROCESS AND PHARMACEUTICAL COMPOSITIONS. Download PDFInfo
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- CH668428A5 CH668428A5 CH2771/86A CH277186A CH668428A5 CH 668428 A5 CH668428 A5 CH 668428A5 CH 2771/86 A CH2771/86 A CH 2771/86A CH 277186 A CH277186 A CH 277186A CH 668428 A5 CH668428 A5 CH 668428A5
- Authority
- CH
- Switzerland
- Prior art keywords
- elution
- carried out
- adsorption
- lactotransferrin
- proteins
- Prior art date
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- 238000000034 method Methods 0.000 title claims description 34
- 239000008194 pharmaceutical composition Substances 0.000 title claims description 8
- 102000014171 Milk Proteins Human genes 0.000 title claims description 4
- 108010011756 Milk Proteins Proteins 0.000 title claims description 4
- 235000021239 milk protein Nutrition 0.000 title claims description 4
- 238000000605 extraction Methods 0.000 title description 4
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 20
- 238000010828 elution Methods 0.000 claims description 18
- 239000005862 Whey Substances 0.000 claims description 12
- 102000007544 Whey Proteins Human genes 0.000 claims description 12
- 108010046377 Whey Proteins Proteins 0.000 claims description 12
- 235000018102 proteins Nutrition 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 229910052742 iron Inorganic materials 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 9
- 238000001179 sorption measurement Methods 0.000 claims description 9
- 150000001768 cations Chemical group 0.000 claims description 5
- 150000002500 ions Chemical class 0.000 claims description 5
- 235000013336 milk Nutrition 0.000 claims description 5
- 239000008267 milk Substances 0.000 claims description 5
- 210000004080 milk Anatomy 0.000 claims description 5
- 239000005018 casein Substances 0.000 claims description 4
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 4
- 235000021240 caseins Nutrition 0.000 claims description 4
- 239000011347 resin Substances 0.000 claims description 4
- 229920005989 resin Polymers 0.000 claims description 4
- 238000000108 ultra-filtration Methods 0.000 claims description 4
- 108060003951 Immunoglobulin Proteins 0.000 claims description 3
- 125000002091 cationic group Chemical group 0.000 claims description 3
- 239000008139 complexing agent Substances 0.000 claims description 3
- 102000018358 immunoglobulin Human genes 0.000 claims description 3
- 229940072221 immunoglobulins Drugs 0.000 claims description 3
- 108010063045 Lactoferrin Proteins 0.000 description 25
- 102000012174 Lactotransferrin Human genes 0.000 description 25
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 239000000243 solution Substances 0.000 description 12
- 239000008363 phosphate buffer Substances 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000011033 desalting Methods 0.000 description 8
- 239000000872 buffer Substances 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 235000020185 raw untreated milk Nutrition 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000002523 gelfiltration Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 229920002684 Sepharose Polymers 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
- 238000011026 diafiltration Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 235000019197 fats Nutrition 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 244000215068 Acacia senegal Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 239000008135 aqueous vehicle Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 229910001447 ferric ion Inorganic materials 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000007923 nasal drop Substances 0.000 description 1
- 229940100662 nasal drops Drugs 0.000 description 1
- 239000002687 nonaqueous vehicle Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 238000009928 pasteurization Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000012465 retentate Substances 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/14—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment
- A23C9/146—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment by ion-exchange
- A23C9/1465—Chromatographic separation of protein or lactose fraction; Adsorption of protein or lactose fraction followed by elution
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/20—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
- A23J1/205—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey from whey, e.g. lactalbumine
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
DESCRIPTION DESCRIPTION
Le présent brevet a pour objet un procédé d'extraction de protéines du lait, notamment celles capables de fixer le fer, son application à la préparation notamment de lactotransferrines, les produits obtenus par le procédé et les compositions pharmaceutiques les renfermant. The present patent relates to a process for extracting milk proteins, in particular those capable of fixing iron, its application to the preparation in particular of lactotransferrins, the products obtained by the process and the pharmaceutical compositions containing them.
Des procédés de préparation de protéines, notamment de lactotransferrines et/ou d'immunoglobulines ont déjà été décrits. On peut citer notamment le brevet français N° 2 505 615 et l'article de Chéron [C. R. Acad. Se. Paris 1284 (14 février 1977)]. Processes for preparing proteins, in particular lactotransferrins and / or immunoglobulins have already been described. Mention may in particular be made of French patent No. 2,505,615 and the article by Chéron [C. R. Acad. Se. Paris 1284 (February 14, 1977)].
Ces procédés ne permettent pas de préparer la lactotransferrine avec une qualité, un rendement et un matériel compatibles avec une préparation industrielle. These processes do not make it possible to prepare lactotransferrin with a quality, a yield and a material compatible with an industrial preparation.
Le procédé selon l'invention permet de préparer des lactopro-téines, notamment la lactotransferrine, en une seule étape d'adsorp-tion-élution sur échangeur d'ions, à pH constant, dans les conditions non dénaturantes et particulièrement douces, avec un matériel léger et industriel et en un temps réduit. The process according to the invention makes it possible to prepare lactoproteins, in particular lactotransferrin, in a single adsorption-elution step on an ion exchanger, at constant pH, in non-denaturing and particularly mild conditions, with a light and industrial equipment and in a reduced time.
C'est ainsi que la présente invention a pour objet un procédé d'extraction des protéines du lait, notamment de celles capables de fixer le fer, à partir d'un lait substantiellement débarrassé de la caséine et des matières grasses, par adsorption sur échangeur d'ions puis élution des protéines adsorbées, caractérisé en ce que l'adsorption et l'élution sont réalisées à pH constant. Thus, the subject of the present invention is a process for extracting milk proteins, in particular those capable of fixing iron, from a milk substantially free of casein and of fat, by adsorption on exchanger ion then elution of the adsorbed proteins, characterized in that the adsorption and the elution are carried out at constant pH.
L'échangeur d'ions utilisé peut être un échangeur d'anions, mais est de préférence un échangeur de cations. Afin d'obtenir une élution plus rapide et plus facile pour éviter la dénaturation des protéines, The ion exchanger used can be an anion exchanger, but is preferably a cation exchanger. In order to get faster and easier elution to avoid protein denaturation,
on utilise de préférence une résine cationique faible, par exemple une résine carboxyméthylique comme celles commercialisées sous les noms CM Trisacryl (IBF) ou CM Sépharose (Pharmacia). preferably a weak cationic resin is used, for example a carboxymethyl resin such as those sold under the names CM Trisacryl (IBF) or CM Sepharose (Pharmacia).
Lorsque l'on utilise une résine cationique, l'adsorption et l'élu-5 tion sont réalisées à un pH inférieur au point isoélectrique de la lactotransferrine, de préférence entre pH 5 et pH 8,5, notamment entre pH 6,5 et 8,3, et tout particulièrement entre pH 7 et pH 8. When using a cationic resin, adsorption and elution are carried out at a pH below the isoelectric point of lactotransferrin, preferably between pH 5 and pH 8.5, in particular between pH 6.5 and 8.3, and especially between pH 7 and pH 8.
Le produit de départ est de préférence un lait cru dont on a préalablement retiré la majeure partie de la caséine et des matières 10 grasses. On utilise par exemple un lactosérum doux ou acide. Le lait ne doit pas avoir été soumis à des opérations détruisant les protéines ou les dégradant, telles que pasteurisation dans des conditions violentes, comme quelques minutes à 90° C par exemple, ou écrémage. Le lait cru peut être, par exemple, décaséiné par précipitation à 15 l'aide d'acide chlorhydrique. The starting product is preferably a raw milk from which most of the casein and fat have previously been removed. For example, a mild or acid whey is used. The milk must not have been subjected to operations destroying the proteins or degrading them, such as pasteurization under violent conditions, such as a few minutes at 90 ° C. for example, or skimming. Raw milk can be, for example, decasinated by precipitation with hydrochloric acid.
La séparation du caillé et du lactosérum est réalisée selon les techniques habituellement utilisées pour les séparations liquide-solide, telles que décantation, centrifugation ou, de préférence, fil-tration. The separation of the curd and the whey is carried out according to the techniques usually used for liquid-solid separations, such as decantation, centrifugation or, preferably, filtration.
20 Dans le cas de la filtration, la membrane choisie ne doit pas de préférence fixer, par adsorption par exemple, les protéines; on utilise par exemple une membrane de nature cellulosique. In the case of filtration, the membrane chosen should preferably not fix the proteins, for example by adsorption; for example, a cellulosic membrane is used.
Dans des conditions préférentielles de mise en œuvre, on part d'un lactosérum concentré. Le lactosérum peut être concentré selon les méthodes classiques comme par évaporation par exemple, dans une proportion variant de 2 à 20 fois par exemple et de préférence d'environ 5 fois. Under preferential processing conditions, we start with a concentrated whey. The whey can be concentrated according to conventional methods such as by evaporation for example, in a proportion varying from 2 to 20 times for example and preferably about 5 times.
Le lactosérum concentré est de préférence dessalé, de manière à diminuer sa force ionique. The concentrated whey is preferably desalted, so as to reduce its ionic strength.
Ces dernières conditions de concentration et force ionique sont avantageusement remplies en une seule étape si l'on effectue une utrafiltration. La nature de l'ultrafiltre doit être choisie de manière à ne pas adsorber les lactoprotéines de manière trop importante, et à les retenir. Le seuil de sélectivité sera par exemple choisi entre 35 10 000 et 70 000, et de préférence entre 25 000 et 50 000, l'ultrafiltre peut se présenter sous forme plane, tubulaire, sous la forme de fibres creuses ou de spirales. Comme ultrafiltres ne fixant pas ou fixant peu les lactoprotéines, notamment la lactotransferrine, on peut citer particulièrement ceux de nature polysulfonique commercialisés sous le nom de Millipore, notamment ceux de référence PSED OHV 10. These latter conditions of concentration and ionic strength are advantageously fulfilled in a single step if one carries out utrafiltration. The nature of the ultrafilter must be chosen so as not to adsorb the lactoproteins too much, and to retain them. The selectivity threshold will for example be chosen between 35 10,000 and 70,000, and preferably between 25,000 and 50,000, the ultrafilter may be in planar, tubular form, in the form of hollow fibers or spirals. As ultrafilters which do not fix or little fixing the lactoproteins, in particular lactotransferrin, mention may be made particularly of those of polysulfonic nature marketed under the name of Millipore, in particular those of reference PSED OHV 10.
Le dessalage et la concentration sont ainsi réalisés par le même dispositif, avec le même appareillage; le dessalage est dans ce cas de préférence effectué par diafiltration (ultrafiltration sous légère surpression) avec Vi à 2 volumes d'eau distillée par exemple. Desalting and concentration are thus carried out by the same device, with the same apparatus; desalting is in this case preferably carried out by diafiltration (ultrafiltration under slight overpressure) with Vi to 2 volumes of distilled water for example.
L'adsorption des lactoprotéines et leur élution sont réalisées en utilisant une même concentration du produit tampon pour conserver le pH choisi, et en modifiant seulement la force ionique à l'aide de chlorure de sodium. Les différentes forces ioniques sont choisies de manière à éluer d'abord les protéines indésirables, puis seulement la 50 protéine désirée. Par exemple, l'élution des protéines capables de fixer le fer, notamment la lactotransferrine, est avantageusement précédée de 1 à 5 élutions de force ionique moindre, et de préférence de 1 à 3 élutions. Le tampon d'élution est de préférence anionique tel un tampon phosphate. The adsorption of the lactoproteins and their elution are carried out by using the same concentration of the buffer product to maintain the chosen pH, and by only modifying the ionic strength using sodium chloride. The different ionic strengths are chosen so as to elute the undesirable proteins first, then only the desired protein. For example, the elution of proteins capable of fixing iron, in particular lactotransferrin, is advantageously preceded by 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.
L'éluat intéressant est ensuite avantageusement dessalé pour éliminer les petites molécules de poids moléculaire inférieur à 1000. Le dessalage est réalisé classiquement, par exemple par tamisage à l'aide d'ultrafiltres Millipore ou par Chromatographie par perméation de 60 gels, G25 par exemple. The interesting eluate is then advantageously desalted to remove small molecules with a molecular weight of less than 1000. Desalting is carried out conventionally, for example by sieving using Millipore ultrafilters or by permeation chromatography of 60 gels, G25 for example .
Le procédé selon la présente invention est particulièrement performant, car un cycle complet de préparation de lactotransferrine pure à partir de lactosérum par ultrafiltration et séparation sur échangeur de cations est réalisé en 9 heures, alors qu'un procédé tel 6J que celui de Chéron ci-dessus cité nécessite 24 heures, soit trois fois plus de temps environ. The process according to the present invention is particularly efficient, since a complete cycle for preparing pure lactotransferrin from whey by ultrafiltration and separation on a cation exchanger is carried out in 9 hours, while a process such as 6J such as that of Chéron above mentioned requires 24 hours, approximately three times more time.
Les lactoprotéines substantiellement pures en solution peuvent alors avantageusement être séchées, notamment par lyophilisation. The substantially pure lactoproteins in solution can then advantageously be dried, in particular by lyophilization.
3 3
668 428 668,428
Les protéines fixant le fer peuvent avantageusement être soumises à l'action d'un complexant, de préférence un chélatant, plus affin qu'elles pour le fer, afin d'augmenter leur capacité en fixation de fer. On utilise par exemple un tampon phosphate EDTA. Proteins fixing iron can advantageously be subjected to the action of a complexing agent, preferably a chelating agent, more refined than they are for iron, in order to increase their capacity in fixing iron. For example, an EDTA phosphate buffer is used.
Le complexant peut être éliminé par dessalage selon les techniques déjà indiquées. The complexing agent can be removed by desalting according to the techniques already indicated.
La présente invention a aussi pour objet les produits obtenus par les procédés ci-dessus décrits, notamment la lactotransferrine et les immunoglobulines. Le procédé ci-dessus décrit est avantageusement utilisé pour leur préparation et l'invention a ainsi également pour objet l'application du procédé décrit ci-dessus à leur préparation. The present invention also relates to the products obtained by the methods described above, in particular lactotransferrin and immunoglobulins. The process described above is advantageously used for their preparation and the invention thus also relates to the application of the process described above to their preparation.
L'intérêt en thérapeutique des lactoprotéines est bien connu, en particulier l'activité bactériostatique de la lactotransferrine. C'est pourquoi la présente invention a enfin pour objet les compositions pharmaceutiques renfermant les produits obtenus par le procédé ci-dessus décrit. En effet, la lactotransferrine obtenue par le procédé de la présente invention a conservé ses propriétés bactériostatiques. The therapeutic interest in lactoproteins is well known, in particular the bacteriostatic activity of lactotransferrin. This is why the present invention finally relates to pharmaceutical compositions containing the products obtained by the process described above. Indeed, the lactotransferrin obtained by the process of the present invention has retained its bacteriostatic properties.
A titre de médicaments, les produits obtenus par le procédé ci-dessus peuvent être incorporés dans des compositions pharmaceutiques destinées à la voie digestive ou parentérale. As medicaments, the products obtained by the above process can be incorporated into pharmaceutical compositions intended for the digestive or parenteral route.
Ces compositions pharmaceutiques peuvent être, par exemple, solides ou liquides et se présenter sous les formes pharmaceutiques couramment utilisées en médecine humaine, comme les comprimés, simples ou dragéifiés, les gélules, les granulés, les suppositoires, les collyres, les solutions nasales. Elles sont préparées selon les méthodes usuelles. Le ou les principes actifs peuvent y être incorporés à des excipients habituellement employés dans ces compositions pharmaceutiques, tels que le talc, la gomme arabique, le lactose, These pharmaceutical compositions can be, for example, solid or liquid and can be in the pharmaceutical forms commonly used in human medicine, such as tablets, simple or coated, capsules, granules, suppositories, eye drops, nasal solutions. They are prepared according to the usual methods. The active ingredient (s) can be incorporated therein into excipients usually used in these pharmaceutical compositions, such as talc, gum arabic, lactose,
l'amidon, le stéarate de magnésium, le beurre de cacao, les véhicules aqueux ou non, les corps gras d'origine animale ou végétale, les dérivés paraffiniques, les glycols, les divers agents mouillants, dispersants ou émulsifiants, les conservateurs. starch, magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, fatty substances of animal or vegetable origin, paraffinic derivatives, glycols, various wetting agents, dispersants or emulsifiers, preservatives.
Les applications des lactotransferrines obtenues par le procédé de l'invention s'étendent, bien entendu, à d'autres secteurs tels que vétérinaires, nutritionnels ou agro-alimentaires. The applications of the lactotransferrins obtained by the process of the invention extend, of course, to other sectors such as veterinary, nutritional or agrifood.
Les exemples qui suivent illustrent la présente invention, sans toutefois la limiter. The examples which follow illustrate the present invention, without however limiting it.
Exemple 1: Extraction de lactotransferrine EXAMPLE 1 Extraction of Lactotransferrin
On ajoute sous agitation 15,8 ml d'acide chlorhydrique à 37-39% à 3,51 de lait cru, laisse précipiter à 40° C pendant une heure, filtre sur membrane cellulosique, soumet les 2100 ml de lactosérum ainsi obtenus à une ultrafiltration sur membrane Millipore de nature polysulfonique à seuil de coupure 25 000 (Référence PSED OHV 10), par diafiltration avec un même volume d'eau distillée, puis concentre à 420 ml. 15.8 ml of 37-39% hydrochloric acid containing 3.51 of raw milk are added with stirring, the mixture is left to precipitate at 40 ° C. for one hour, filtered on a cellulose membrane, and the 2100 ml of whey thus obtained is subjected to ultrafiltration on a Millipore polysulfonic membrane with a cutoff threshold of 25,000 (Reference PSED OHV 10), by diafiltration with the same volume of distilled water, then concentrated to 420 ml.
On ajuste à 7,0 le pH de la solution concentrée à l'aide de soude et ajuste la force ionique à 0,15M à l'aide de chlorure de sodium (en mesurant au conductimètre). 400 ml de solution sont passés, au débit de 3 ml/min, sur une colonne de 25 mm de diamètre interne remplie de 100 ml de CM Sépharose CL 6B (Pharmacia) préalablement équilibré dans un tampon phosphate 0,15M pH 7,0, en effectuant les élutions au débit de 3 ml/min avec un tampon phosphate 0,1M pH 7,0 dont la force ionique est ajustée à l'aide de chlorure de sodium, selon le gradient suivant: The pH of the concentrated solution is adjusted to 7.0 using sodium hydroxide and the ionic strength is adjusted to 0.15 M using sodium chloride (by measuring with a conductimeter). 400 ml of solution are passed, at a flow rate of 3 ml / min, over a column 25 mm in internal diameter filled with 100 ml of CM Sepharose CL 6B (Pharmacia) previously equilibrated in a 0.15 M phosphate buffer pH 7.0, by carrying out the elutions at the flow rate of 3 ml / min with a 0.1M phosphate buffer pH 7.0 whose ionic strength is adjusted using sodium chloride, according to the following gradient:
270 ml à 0,15M 270 ml at 0.15M
145 ml à 0,25M 145 ml at 0.25M
145 ml à 0,30M. 145 ml at 0.30M.
La lactotransferrine est éluée avec 430 ml à 0,4M. On opère un dessalage sur gel G25 Pharmacia, lyophilise la solution obtenue et obtient 84 mg de lactotransferrine. The lactotransferrin is eluted with 430 ml at 0.4M. Desalting is carried out on a G25 Pharmacia gel, lyophilizes the solution obtained and obtains 84 mg of lactotransferrin.
Exemple 2: Extraction de lactotransferrine EXAMPLE 2 Extraction of Lactotransferrin
On opère comme indiqué à l'exemple 1 à partir de 1,51 de lait cru. The operation is carried out as indicated in Example 1 from 1.51 of raw milk.
150 ml de solution sont passés sur colonne au débit de 1 ml/min. On effectue une élution avec 150 ml de tampon phosphate 0,15M, 150 ml of solution are passed through a column at a flow rate of 1 ml / min. An elution is carried out with 150 ml of 0.15 M phosphate buffer,
pH 7,0 de force ionique 0,25M, puis élue la lactotransferrine avec 80 ml de tampon pH 7,0 de force ionique 0,43M. pH 7.0 of 0.25M ionic strength, then elute the lactotransferrin with 80 ml of buffer pH 7.0 of 0.43M ionic strength.
Après dessalage et lyophilisation, on obtient 40 mg de lactotransferrine. After desalting and lyophilization, 40 mg of lactotransferrin are obtained.
Exemple 3: Extraction de lactotransferrine EXAMPLE 3 Extraction of Lactotransferrin
Le lactosérum est préparé et déposé sur l'échangeur cationique faible comme indiqué à l'exemple 1. The whey is prepared and deposited on the weak cation exchanger as indicated in Example 1.
On pratique alors un lavage de la colonne et de l'échangeur avec> 200 ml de tampon phosphate 0,1M pH 7,0 dont la force ionique est ajustée à 0,15M à l'aide de chlorure de sodium. La lactotransferrine est ensuite éluée avec 100 ml de même tampon dont la force ionique est ajustée à 1M à l'aide de chlorure de sodium. Le dessalage est effectué de la même façon que précédemment (filtration sur gel G25 Pharmacia). Après lyophilisation, le rendement de l'opération est d'environ 90 mg de lactotransferrine pour 3,5 1 de lait. The column and the exchanger are then washed with> 200 ml of 0.1M phosphate buffer, pH 7.0, the ionic strength of which is adjusted to 0.15M using sodium chloride. The lactotransferrin is then eluted with 100 ml of the same buffer, the ionic strength of which is adjusted to 1M using sodium chloride. Desalting is carried out in the same way as above (filtration on G25 Pharmacia gel). After lyophilization, the yield of the operation is approximately 90 mg of lactotransferrin per 3.5 l of milk.
Exemple 4: Example 4:
Le lactosérum est préparé comme dans les exemples précédents. On ajuste son pH à 7,0 à l'aide de soude. On ajoute ensuite la solution d'AZARI et BAUGH à raison de 1 ml par litre de lactosérum. The whey is prepared as in the previous examples. Its pH is adjusted to 7.0 using sodium hydroxide. Then add the AZARI and BAUGH solution at a rate of 1 ml per liter of whey.
On laisse la saturation de la lactotransferrine en ions ferriques se réaliser à température ambiante pendant 2 heures. La solution est ensuite centrifugée puis ultrafiltrée, comme dans les exemples précédents. The lactotransferrin saturation with ferric ions is allowed to take place at room temperature for 2 hours. The solution is then centrifuged and then ultrafiltered, as in the previous examples.
On passe 400 ml du rétentat obtenu sur 100 ml d'échangeur cationique faible puis, après un lavage avec 200 ml de tampon phosphate 0,15M pH 7,0, la lactotransferrine saturée est éluée avec 100 ml du même tampon dont la force ionique est ajustée à 1M à l'aide de chlorure de sodium. 400 ml of the retentate obtained are passed over 100 ml of weak cation exchanger and then, after washing with 200 ml of 0.15 M phosphate buffer, pH 7.0, the saturated lactotransferrin is eluted with 100 ml of the same buffer, the ionic strength of which is adjusted to 1M using sodium chloride.
La préparation est dessalée par filtration sur gel (G25) puis lyophilisée. The preparation is desalted by gel filtration (G25) and then lyophilized.
On dissout la lactotransferrine saturée, dessalée dans un tampon phosphate/citrate 0,05M pH 4,5, à raison de 1 mg par ml de tampon. The saturated, desalted lactotransferrin is dissolved in a 0.05M phosphate / citrate buffer pH 4.5, at a rate of 1 mg per ml of buffer.
La désaturation s'effectue pendant 12 heures à +4° C. La solution de lactotransferrine désaturée est ensuite dessalée par filtration sur gel (G25) puis lyophilisée. Desaturation is carried out for 12 hours at + 4 ° C. The solution of desaturated lactotransferrin is then desalted by gel filtration (G25) and then lyophilized.
Composition de la solution d'AZARI et BAUGH Composition of the AZARI and BAUGH solution
Préparer une solution 0,1M en citrate de sodium et 0,1M en bicarbonate de sodium. Prepare a 0.1M sodium citrate and 0.1M sodium bicarbonate solution.
A 100 ml de cette préparation, ajouter 232 mg de chlorure ferri-que-6H20. To 100 ml of this preparation, add 232 mg of ferri-que-6H20 chloride.
Exemple 5: Example 5:
Du lactosérum est préparé et concentré comme dans les exemples précédents. On ajuste à 7,0 le pH de la solution concentrée à l'aide de soude. Whey is prepared and concentrated as in the previous examples. The pH of the concentrated solution is adjusted to 7.0 with sodium hydroxide.
Dix litres de préparation sont passés au débit de 6 litres à l'heure sur une colonne de 10 cm de diamètre remplie de 600 ml de CM Se-pharose Fast-Flow (Pharmacia) préalablement équilibré dans un tampon phosphate 0,15M pH 7,0. Ten liters of preparation are passed at a flow rate of 6 liters per hour on a column 10 cm in diameter filled with 600 ml of CM Se-pharose Fast-Flow (Pharmacia) previously balanced in 0.15M phosphate buffer pH 7, 0.
Après le dépôt, on lave la colonne avec un litre de tampon phosphate 0,15M pH 7,0 puis avec un litre du même tampon dont la force ionique est ajustée à 0,3M à l'aide de chlorure de sodium. After the deposition, the column is washed with one liter of 0.15 M phosphate buffer, pH 7.0, then with one liter of the same buffer, the ionic strength of which is adjusted to 0.3 M using sodium chloride.
La lactotransferrine est ensuite éluée avec 600 ml du même tampon dont la force ionique est ajustée cette fois à 1M. The lactotransferrin is then eluted with 600 ml of the same buffer, the ionic strength of which is adjusted this time to 1M.
On opère un dessalage par filtration sur gel (G25) puis on lyophilise la préparation. On obtient environ 30 mg de lactotransferrine par litre de substrat. Desalting is carried out by gel filtration (G25) and the preparation is lyophilized. About 30 mg of lactotransferrin are obtained per liter of substrate.
Exemple 6: Composition pharmaceutique Example 6 Pharmaceutical Composition
On a préparé des gouttes nasales ayant la composition suivante: Nasal drops having the following composition were prepared:
— Lactotransferrine 5 mg - Lactotransferrin 5 mg
— Stabilisant 250 mg - Stabilizer 250 mg
— Eau distillée q.s.p. 5 ml - Distilled water q.s.p. 5 ml
5 5
10 10
15 15
20 20
25 25
30 30
35 35
40 40
45 45
50 50
55 55
60 60
65 65
R R
Claims (14)
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8510649A FR2584727B1 (en) | 1985-07-11 | 1985-07-11 | PROCESS FOR EXTRACTING MILK PROTEINS, PRODUCTS, APPLICATION OF THE PROCESS, AND PHARMACEUTICAL COMPOSITIONS |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CH668428A5 true CH668428A5 (en) | 1988-12-30 |
Family
ID=9321210
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CH2771/86A CH668428A5 (en) | 1985-07-11 | 1986-07-10 | PROCESS FOR THE EXTRACTION OF MILK PROTEINS, PRODUCTS, APPLICATION OF THE PROCESS AND PHARMACEUTICAL COMPOSITIONS. |
Country Status (11)
| Country | Link |
|---|---|
| JP (1) | JPS6219523A (en) |
| BE (1) | BE905087A (en) |
| CH (1) | CH668428A5 (en) |
| DE (1) | DE3623474C2 (en) |
| DK (1) | DK327386A (en) |
| FR (1) | FR2584727B1 (en) |
| GB (1) | GB2179947B (en) |
| IT (1) | IT1195855B (en) |
| LU (1) | LU86508A1 (en) |
| NL (1) | NL8601814A (en) |
| SE (1) | SE8602877L (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0621077B2 (en) * | 1986-07-15 | 1994-03-23 | 雪印乳業株式会社 | Hematopoietic agent |
| IE61701B1 (en) * | 1986-07-17 | 1994-11-30 | Morinaga Milk Industry Co Ltd | Process for producing bovine lactoferrin in high purity |
| FR2613725A1 (en) * | 1987-04-07 | 1988-10-14 | Agronomique Inst Nat Rech | Process for the production of active lactoperoxydase and lactoferrin from whey and the substances produced by this process |
| JPH0725800B2 (en) * | 1987-04-10 | 1995-03-22 | 雪印乳業株式会社 | Method for separating and purifying lactoferrin from milk using sulfate esterification product |
| SE458818B (en) * | 1987-11-27 | 1989-05-16 | Svenska Mejeriernas Riksforeni | PROCEDURE FOR EXTRACTION OF PURE FRACTIONS OF LACTOPEROXIDAS AND LACTOFERRIN FROM MILK SERUM |
| FR2626472B1 (en) * | 1988-02-02 | 1991-06-14 | Roussel Uclaf | USE OF MILK PROTEINS FOR THE MANUFACTURE OF AN ANTI-VIRAL DRUG |
| FR2631785A1 (en) * | 1988-05-27 | 1989-12-01 | Agronomique Inst Nat Rech | METHOD FOR FRACTIONING HUMAN MILK PROTEINS, DRIVING PRODUCTION, IN PARTICULAR LACTOFERRIN AND (ALPHA) -LACTALBUMIN, AND PRODUCTS OBTAINED |
| FR2648321B1 (en) * | 1989-05-12 | 1992-01-17 | Bio Serae Lab | PROCESS OF TREATING A NON-LIQUID FOOD PRODUCT TO ENSURE MICROBIAL DECONTAMINATION, APPLICATIONS IN PARTICULAR TO CHEESE AND MOTHER PREPARATION FOR IMPLEMENTING SAID TREATMENT |
| ATE287415T1 (en) * | 1990-07-13 | 2005-02-15 | Gropep Ltd | GROWTH-PROMOTING ACTIVE INGREDIENT |
| JP3035833B2 (en) * | 1991-01-21 | 2000-04-24 | 雪印乳業株式会社 | Method for producing sialic acids-containing composition |
| JP2961625B2 (en) * | 1991-01-21 | 1999-10-12 | 雪印乳業株式会社 | Method for producing a composition having a high content of α-lactalbumin |
| EP0595993A4 (en) * | 1991-07-25 | 1994-08-17 | Commw Scient Ind Res Org | Isolation of charged particles from fluids |
| CA2162671A1 (en) * | 1993-05-11 | 1994-11-24 | Daniel E. Beidler | Separation of anti-metal chelate antibodies |
| DK0876106T3 (en) | 1996-01-26 | 2004-08-16 | Univ Massey | Process for separating and recovering proteins from a protein solution |
| US6268487B1 (en) | 1996-05-13 | 2001-07-31 | Genzyme Transgenics Corporation | Purification of biologically active peptides from milk |
| AU738888B2 (en) | 1997-05-29 | 2001-09-27 | Agresearch Limited | Processes for production of immunoglobulin A in milk |
| AU1313401A (en) * | 1999-10-26 | 2001-05-08 | Fonterra Co-Operative Group Limited | Method of obtaining immunoglobulins from colostrum and dairy sources |
| JP2001231510A (en) * | 2000-02-22 | 2001-08-28 | Snow Brand Milk Prod Co Ltd | Seafood-paste |
| CN101072580B (en) | 2004-10-06 | 2012-08-22 | 农业生物技术有限公司 | Antibody production method |
| AU2013204850B2 (en) * | 2012-10-08 | 2015-06-25 | Murray Goulburn Co-Operative Co. Limited | Improved process for purifying milk proteins and products thereof |
| AU2013204858B2 (en) * | 2012-10-08 | 2015-06-25 | Saputo Dairy Australia Pty Limited | Improved process for purifying milk proteins and products thereof |
| CN104109204B (en) * | 2013-04-16 | 2017-11-07 | 武汉禾元生物科技股份有限公司 | A kind of method that restructuring lactoferrin is isolated and purified from rice paddy seed |
-
1985
- 1985-07-11 FR FR8510649A patent/FR2584727B1/en not_active Expired
-
1986
- 1986-06-27 SE SE8602877A patent/SE8602877L/en not_active Application Discontinuation
- 1986-07-10 GB GB8616819A patent/GB2179947B/en not_active Expired
- 1986-07-10 IT IT48254/86A patent/IT1195855B/en active
- 1986-07-10 LU LU86508A patent/LU86508A1/en unknown
- 1986-07-10 CH CH2771/86A patent/CH668428A5/en not_active IP Right Cessation
- 1986-07-10 BE BE0/216905A patent/BE905087A/en not_active IP Right Cessation
- 1986-07-10 NL NL8601814A patent/NL8601814A/en not_active Application Discontinuation
- 1986-07-10 DK DK327386A patent/DK327386A/en not_active Application Discontinuation
- 1986-07-11 DE DE3623474A patent/DE3623474C2/en not_active Expired - Fee Related
- 1986-07-11 JP JP61162200A patent/JPS6219523A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| BE905087A (en) | 1987-01-12 |
| GB2179947A (en) | 1987-03-18 |
| FR2584727A1 (en) | 1987-01-16 |
| FR2584727B1 (en) | 1988-06-17 |
| DE3623474C2 (en) | 1995-09-21 |
| IT8648254A0 (en) | 1986-07-10 |
| LU86508A1 (en) | 1987-02-04 |
| SE8602877D0 (en) | 1986-06-27 |
| JPS6219523A (en) | 1987-01-28 |
| SE8602877L (en) | 1987-01-12 |
| GB2179947B (en) | 1989-07-12 |
| DE3623474A1 (en) | 1987-01-15 |
| GB8616819D0 (en) | 1986-08-20 |
| DK327386D0 (en) | 1986-07-10 |
| DK327386A (en) | 1987-01-12 |
| NL8601814A (en) | 1987-02-02 |
| IT1195855B (en) | 1988-10-27 |
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