MXPA96003369A - Isolation of lactoferrina from le - Google Patents

Abstract

The invention relates to methods for the purification of human lactoferrin from milk, especially milk from non-human species, and for the separation of human lactoferrin from unwanted macromolecular species present in milk, including the separation of lactoferrin species no huma

Description

ISOLATION OF LACTOFERRINE FROM MILK FIELD OF THE INVENTION The invention relates to the purification of lactoferrin from milk, particularly the purification of human lactoferrin from the milk of transgenic non-human animals expressing a human lactoferrin polypeptide encoded by a transgene. BACKGROUND Recent advances in the field of molecular biology allow the production of transgenic animals (ie, non-human animals that contain an exogenous DNA sequence in the genome of the germ line and somatic cells introduced by means of human intervention). The differences in the regulation of these foreign genes in different cell types makes it possible to promote the differential expression of the foreign gene in a preselected tissue, such as the mammary gland, for ease of isolation of the protein encoded by the foreign gene, for a desired activity of the foreign gene product in the selected tissues, or for other reasons. An advantage of transgenic animals and the expression of the differential gene is the isolation of important proteins in large quantities, especially by means of economic purification methods. Such proteins are typically exogenous to the transgenic animal and may comprise pharmaceuticals, food additives, nutritional supplements, and the like. However, exogenous proteins are preferably expressed in tissues analogous to those in which they are naturally expressed. For example, exogenous milk proteins (e.g., lactoferrin) are preferentially expressed in milk-forming tissues in the transgenic animal. As a result, difficult problems of isolation arise since the exogenous protein is often expressed in body tissue or fluid containing an endogenous complement protein (if it exists), and possibly other unwanted contaminant species that may have physical properties. very similar chemistries. In addition, many exogenous proteins must be substantially purified from other species, often purified by homogeneity, before being used as pharmaceutical or food additives. For example, the production of transgenic bovine species containing a transgene encoding a human lactoferrin polypeptide directed at expression in mammary secretion cells is described in O91 / 08216, is incorporated herein by reference. The purification of human lactoferrin (hLF) from a transgenic animal that contains a functional endogenous bovine lactoferrin gene (bLF) and a transgene that encodes the expression of hLF is complicated in the presence of endogenous bLF, which has physico-chemical properties similar to those of human lactoferrin. Even in a transgenic bovine lacking a functional endogenous bLF gene (e.g., as a result of targeting the homologous gene to functionally interrupt alleles of endogenous bLF), it is often desirable and / or necessary to purify hLF encoded by transgene from other biological macromolecules and contaminating species. Since hLF has potential pharmaceutical uses and can be incorporated into human food products as a nutritional supplement, uses that typically require a highly purified hLF, it is imperative that methods are developed to purify hLF from milk, especially milk or fractions. of milk of non-human transgenic animals such as bovine species. Human lactoferrin is a single chain glycoprotein that binds ferric ions. Segregated by the exocrine glands (Mason et al. (1978) J. Clin. Path 31: 316; Tennuovo et al. (1986) Infect. Immunol. 51:49) and contained in neutrophilic leukocyte granules (Mason et al. (1969) J Ex. Med. 130: 643), this protein functions as part of a non-specific defense system of the host by inhibiting the growth of a different spectrum of bacteria. HLF exhibits a bacteriostatic effect by chelating the available iron in the medium, making this essential metal inaccessible to microorganisms (Bullen et al. (1972) Brit. Med. J. 1: 69; Griffiths et al. (1977) Infect. Immunol. 15: 396; Spik et al. (1978) Immunoloay 8: 663 Stewart et al. (1984) Int. J. Biochen-16: 1043). The bacteriostatic effect can be blocked if the ferric ions present in excess to those necessary to saturate the binding sites of the hLF. Lactoferrin is a major protein in human milk (present at a concentration of approximately 1.5mg / ml) and may play a role in the absorption of dietary iron by the small intestine. Essentially, all the iron present in human breast milk is reported to be linked to hLF and is taken very efficiently by the intestine compared to free iron in infant formula (Hide et al. (1981) Arch. Dis. Child. 56: 172). It has been postulated that the efficient intake of iron bound to hLF in humans is due to a receptor in the duodenum (Cox et al. (1979) Biochim, Biophys, Acta 588: 120). Specific lactoferrin receptors have been reported in the mucosal cells of the small intestine of human fetuses (Kawakami and Lonnerdal (1991) Am. J. Physiol. 261: G841). The hLF of human colostrum is commercially available (Calbiochem, La Jolla, California and other vendors) as a lyophilisate for research applications in small amounts (10 mg and 25 mg vials). The amino acid sequence of hLF has been reported (Metz-Boutigue et al. (1984) Eur. J. Biochem. 1451: 659), and O91 / 08216 reports a hLF sequence that has some sequence inconsistencies with the previous report from Metz-Boutigue et al. hLF comprises two fields of action, each comprising an iron binding site and a N-linked glycosylation site. These fields of action show homology to each other, consistent with an ancestral gene duplication and a fusion event. HLF also shares extensive sequence homology with other members of the transferrin family (Metz-Boutigue et al. (1984) op. Cit ..- Pentecost et al. (1987) J. Biol. Chem. 262: 10134). A partial cDNA sequence for the neutrophil hLF was published by Rado et al. (1987) Blood 70: 989), which is more than 98% consistent with the sequence identity compared to the amino acid sequence determined by direct amino acid sequence from hLF of human milk. The structures of the iron-saturated and iron-free forms of human lactoferrin are have reported (Anderson et al. (1989) J. Mol. Biol. 209: 711; Anderson et al. (1990) Nature 344: 784). Protocols have been reported to purify lactoferrin from milk. U.S. Patent 4,436,658 describes the isolation of bovine lactoferrin from nonfat and casein-free bovine whey. Briefly, the serum is contacted with silica in a slightly basic medium at pH 7.7-8.8, the lactoferrin is adsorbed and subsequently eluted with 0.5M NaCl / O.lN acetic acid. U.S. Patent 4,791,193 and European Patent Application No. EP 0 253 395 to okonogi et al. Similarly report a method wherein bovine milk is contacted with carboxymethyl groups of a mildly acidic cation exchange resin and the Absorbed lactoferrin is eluted with a gradient of 10 percent NaCl. In U.S. Patent 4,668,771, bLF is separated from bovine milk using a monoclonal antibody bound to an insoluble carrier. In WO89 / 04608 a process for obtaining fractions of bovine lactoperoxidase and bLF from bovine whey is described, - the whey is microfiltered and passes through a strong cation exchanger, pH 6.5, at a rate of high flow for the adsorption of lactoperoxidase with bLF with a 0.1-0.4M solution and a of 0.5-2.0M NaCl, respectively. U.S. Patent 4,997,914 discloses the isolation of hLF from human or whole milk colostrum, - the sample containing lactoferrin is contacted with a sulfuric ester of a degraded polysaccharide to bind hLF, followed by elution with an aqueous solution of 0.4 -1.5 of NaCl. The scientific literature also reports protocols for the isolation of lactoferrin from milk. Some of these involve the isolation of LF from a natural source through the use of ion exchange chromatography followed by salt elution. Querinjean et al. (1971) Eur. J. Biochem. 20: 420, reports the isolation of hLF from human milk on a CM Sephadex C-50 followed by elution with 0.33M NaCl. Johannson (1969) Acta Chem. Scand. 23: 683 used the CM Sephadex C-50 for the purification of the LF, and Johannson et al. (1958) Nature 181: 996 reports the use of calcium phosphate for the purification of the LF. Torres and others (1979) Biochem. Biphys. Acta 576: 385 reports the isolation of lactoferrin from guinea pig milk. The milk is preheated by centrifugation to remove fats and sediment casein. A Whatman CM-52 column was used, and lactoferrin was eluted with 0.5M NaCl / 5mM sodium phosphate, pH 7.5. Roberts and Boursnell (1975) Jour. Of Reproductive Fertility 42: 579, reports the lactoferrin isolated from non-fat sow milk. CM-Sephadex was added to a ferrous sulfate precipitate of milk ammonia, and the bound lactoferrin was eluted with 0.5M NaCl / 20mM phosphate at a pH of 7 followed by a second fractionation of CM-Sephadex from the which lactoferrin is eluted with 0.4M NaCl. Zagulski et al. (1979) Prace i Materialy Zootechniczne 20: 87, reports bovine lactoferrin separated from bovine milk. Skimmed bovine milk was mixed with CM-Sephadex C-50, and lactoferrin was eluted from the column with 0.5M sodium chloride / 0.02M sodium phosphate at a pH of 7. Moguilevsky et al. (1975) Biochem. J. 229: 353, reports lactoferrin isolated from human milk, by the use of CM-Sephadex chromatography and elution with IM sodium chloride. Ekstrand and Bjorck (1986) Jour. Of chromatography 358: 429, report lactoferrin isolated from human colostrum and from bovine milk. The human or bovine skim milk was acidified. It was adjusted to a pH of 7.8 and applied to a Mono S TM column. Human or bovine lactoferrin was eluted with a continuous salt gradient of 0.% NaCl The purification of human lactoferrin from bovine lactoferrin was not reported. Foley and Bates (1987) Anal. Biochem. 162: 296, report the isolation of lactoferrin from human colostrum serum. He The serum was mixed with a soft ion exchange resin (cellulose phosphate) and the proteins were eluted with a progressive salt and a pH gradient. Lactoferrin was eluted with 0.25M NaCl / 0.2M sodium phosphate at a pH of 7.5. In addition, Yoshida and Ye-Xiuyun (1991) J. Dairy Sci. 74: 1439, exposed the isolation of lactoferrin by ion exchange in a carboxymethyl cation resin using 0.05M of buffered phosphate at a pH of 7.7 with a gradient. Linear 0-0.55M NaCl. The carboxymethyl-Toyopearl column absorbed only lactoperoxidase and lactoferrin from the albumin fraction of the bovine milk acid serum. Lactoferrin was eluted between 0.4-0.55M NaCl and separated into two components; lactoferrin A and lactoferrin B. Other methods were also reported, including affinity chromatography. For example, in Kawakami et al. (1987) J. Daury Sci. 70: 752, affinity chromatography of LF is reported with human or bovine lactoferrin monoclonal antibodies. Human lactoferrin was separated from human colostrum and bovine lactoferrin was separated from bovine milk or cheese whey. (See also U.S. Patent 4,668,771, cited above). In Hutchens et al. (1989) Clin. Chem. 35: 1928, lactoferrin was separated from the urine of premature infants fed human milk with A- DNA of a single chain in an affinity column. Additionally, Chen and Wang (1991) J. Food Sci. 56: 701 reported a process that combines affinity chromatography with membrane filtration to separate lactoferrin from bovine cheese whey using heparin-Sepharose to bind lactoferrin. The cheese whey was diluted with a binding buffer and added to the heparin-Sepharose material. The mixture was microfiltered, and the lactoferrin was eluted with 5 mM veronal hydrochloride / 0.6 M NaCl at a pH of 7.4. Bezwoda et al. (1986) Clin Chem. Acta 157: 89 reports the use of Cibacron Blue F3GA resin for the purification of LF. Ferritin (Pahud et al. (1976) Protides Biol Fluids, 23: 571) and heparin (Blackberg (1980) FEBS Lett. 109: 180) have also been reported for purification of milk. Thus, there is a need in the art for methods of purifying human lactoferrin from milk, particularly from the milk of non-human transgenic animals, such as bovine species, containing human lactoferrin encoded by a transgene. It is an object of the present invention to provide methods and compositions for the economical and efficient purification of human lactoferrin from milk, such as bovine milk, to be used as a pharmaceutical or food additive. The present invention It satisfies these and other needs. It is also an object of the present invention to provide human lactoferrin compositions with a purity of about 98% or more. The references discussed herein are provided only for disclosure prior to the filing date of the present application. Nothing in the present should be considered as an admission that the inventors are not authorized to precede such disclosure by virtue of the prior invention. SUMMARY OF THE INVENTION The present invention provides an efficient and effective method for the purification of human lactoferrin from milk, especially for the purification of human lactoferrin from bovine milk produced by transgenic bovine species containing a transgene of human lactoferrin. The human lactoferrin encoded by the transgene is substantially purified from other milk proteins in the milk of the transgenic cows, and is preferably substantially separated from the endogenous bovine lactoferrin, if present in the milk. The invention provides methods for separating human lactoferrin, including human lactoferrin produced by the expression of a transgene encoding a Recombinant human lactoferrin (rhLF), as well as other related lactoferrin species from milk, typically from bovine milk. Such other related lactoferrin species may include truncated hLF, variants of the amino acid sequence (polymorphic variants or muteins) of hLF, hLF species comprising additional residues. The invention also provides methods that allow the purification of human lactoferrin (including rhLF) from bovine lactoferrin, ovine lactoferrin, goat lactoferrin, mouse lactoferrin, and porcine lactoferrin. In general, milk or a fraction of milk containing hLF is contacted with a strong cation exchange resin (eg, Sepharose Sepharose ™) in the presence of a relatively high ionic strength (0.2M a 0. 5M NaCl or KC1, preferably 0.4M NaCl or KC1) to avoid the binding of proteins other than lactoferrin and other substances to the strong cation exchange resin and to reduce the electrostatic interactions of lactoferrin with other proteins (eg, caseins) or substances (eg, lipopolysaccharides), and to release lactoferrin from the complexes. The strong cation exchange resin containing the lactoferin bond is separated from the unbound compounds in the milk or milk fraction, typically by centrifugation or sedimentation followed by rinsing in discontinuous form and / or spilling the resin into a column and rinsing the beads with a regulator having an approximately equal or lower salt concentration. The binding of lactoferrin to the cation exchange resin is eluted with a gradient of typically regulated, aqueous NaCl or KC1 (eg, a linear gradient of 0-1M NaCl in 20mM sodium phosphate, pH 7.5) or by discontinuous elution or stepwise elution with a NaCl or KCl solution, preferably regulated, aqueous, of 0.4 M or more, preferably of at least 0.5 M NaCl or KCl. By selecting the appropriate elution conditions, human lactoferrin can be substantially purified from bovine milk and substantially separated from bovine lactoferrin by an efficient method. In one aspect of the invention, human lactoferrin (e.g., rhLF) is further purified from an endogenous lactoferrin (e.g., bLF) by the further subsequent step of rechromatography in a strong cation exchanger, such as Fast Flow. of S Sepharose TM, with a stepwise graded salt or elution ** to separate human lactoferrin from the remaining clues of endogenous non-human lactoferrin species (eg, bLF) and / or may include optionally affinity chromatography with a concanavalin A resin to further separate the human lactoferrin from the bLF, the bLF being bound more strongly with the resin A of Con than with the hLF. In a method of the invention, a limiting amount of a strong cation exchange resin (e.g., an amount less than that necessary to saturably bind essentially all of the lactoferrin in the sample) is contacted with milk or a Milk fraction (eg, whey) under aqueous conditions (eg, by adding the resin directly to the milk or milk fraction) by which stronger cationic proteins, such as lactoferrin, bind competitively and preferentially to the limiting amount of the cation exchange resin present. With a limiting amount of the cation exchange resin we mean an amount that is just enough to bind at least essentially all (eg, 99 percent) the molecules of an object (eg human) lactoferrin species in a resistance of predetermined salt of the milk or fraction of milk. This limits the amount of the strong cation resin to be used to optimize the selectivity of the lactoferrin linkage. The limiting amount of the strong cation exchange resin with bound protein is separated from the rest of the milk or milk fraction, typically by centrifugation or sedimentation followed by discontinuous rinsing and / or pouring of the resin into a column followed by column rinsing of the resin and bound proteins. The lactoferrin linkage with the resin is eluted by a high salt buffer (i.e., a NaCl or KC1 concentration greater than 0.4M) or a salt gradient having a maximum salt concentration of at least about 0.5M NaCl or KC1). In one variation, ionic species, such as NaCl or KC1, are added to whole milk, processed milk, or a milk fraction before contacting the milk or milk fraction with a strong exchange resin. of cation. Typically, the salt (or a salt solution) is added to the milk or milk fraction to conduct the final salt concentration to about 0.2M to 0.5M, more preferably to about 0.4M in NaCl or KC1, forming a high ionic strength milk solution. The high ionic strength milk solution is contacted with a strong cation exchange resin under the binding conditions and the resin containing the bound milk protein (s) is separated from the non-bound components. linked in the rest of the solution high ionic strength milk, typically by centrifugation or sedimentation followed by rinsing in discontinuous form and / or by spillage of the resin mixture of strong cation exchange in a column, and removal of the rest of the high-strength milk solution ion when removing the liquid from the column and / or by rinsing the resin column with a rinse regulator having an ionic strength approximately equal to or lower than the high ionic strength milk solution. The linkage of lactoferrin to the strong cation exchange resin is eluted by a high salt buffer (ie, a NaCl or KCl concentration greater than 0.4M) or a salt gradient having a maximum salt concentration of at least approximately 0.5M NaCl or KCl). In an optional variation, a detergent, such as a non-ionic detergent (e.g., Tween-20) may be added to the milk or milk fraction to reduce undesirable hydrophobic interactions between macromolecules that would reduce the efficiency of lactoferrin purification. The milk or milk fraction can also be eluted substantially, typically to a final salt concentration of about 0.2M to 0.5M NaCl or KCl, preferably 0.4M NaCl or KCl, in order to further reduce intermolecular interactions not desired that can reduce the production and / or purity of recovered lactoferrin. All publications and patent applications herein are incorporated for reference to the same extent that each publication or individual patent application would be incorporated for reference in a specific and individual manner. Brief Description of the Drawings Figure 1 shows the differential elution profiles of the hLF (panel A) and the bLF (panel B) from a resi «na strong exchange of cati • ** ** n, Mono STM by a linear salt gradient. Figure 2 shows the elution profiles of hLF (panel A) and bLF (panel B) from a strong cation exchange resin, Mono S ™ by step elution. Figure 3 shows the resolution of hLF from bLF in a strong cation exchange resin by elution with a linear salt gradient (panel A) and stepped elution (panel B). Figure 4 shows specific radioimmunoassays for hLF and bLF in the fractions eluted by elution with a linear salt gradient (panel A) and stepped elution (panel B). Figure 5 shows the chromatographic resolution of hLF from bLF in bovine milk with hLF omitted (panel B) chromatographed on a Mono STM column with a linear salt gradient elution. Panel A shows control bovine milk (without omitting). Figure 6 shows the chromatographic resolution of hLF from bLF in bovine milk with hLF omitted (panel B) chromatographed on a Mono S column with stepwise elution of salt. Panel A shows control bovine milk (without omitting). Figure 7 shows the relationship between the amount of hLF bound to a Mono S TM column and the concentration of NaCl at which it is observed that hLF elution is initiated when a linear salt gradient is applied. Figure 8 shows the profiles for the elution of hLF (A) and bLF (B) from a PF Sepharose FF column. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary experience in the subject matter to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or examination of the present invention, preferred methods and materials are described. For the purposes of this invention, the following terms are defined below. The term "naturally occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that occurs in an organism (including viruses) that can be separated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. As used herein, "substantially pure" means that an object species is the current predominant species (ie, on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the target species comprise at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 to 90 percent of all macromolecular species present in the composition. More preferably, the target species are purified to essential homogeneity (contaminating species can not be detected by conventional detection methods) wherein the composition consists essentially in a single macromolecular species. As used herein the term "enriched" refers to a composition or fraction in which an object species has been partially purified in such a way that, on a molar proportion basis, at least approximately 10 percent of one or more naturally occurring pollutant species has been removed. For example, a milk sample of a transgenic bovine expressing human lactoferrin can be enriched in human lactoferrin by selectively removing the caseins by acid precipitation (for example, the serum fraction is enriched by this of human lactoferrin). As used herein, "human lactoferrin" comprises a polypeptide having an amino acid sequence substantially as described by Metz-Boutigue et al. (1984) Eur. J. Biochem. 1451: 659, observing the sequential inconsistencies identified in PCT publication WO91 / 08216 and other published proteins and DNA sequences. The term human lactoferrin also includes naturally occurring human allelic variants either (partially) proteolyzed or not ("naturally occurring human lactoferrin") and amino acid sequence variants that have been modified by insertion, substitution, or deletion of one or more amino acids compared to a naturally occurring species of human lactoferrin and which has a higher degree of sequential identity when aligned optimally (and with interstices, if necessary) with a naturally occurring at least 50% amino acid sequence of human lactoferrin. contiguous amino acids, than other naturally occurring polypeptide species of at least 50 contiguous amino acids. Human lactoferrin also includes recombinantly encoded human lactoferrin ("rhLF") expressed in a transgenic non-human animal, such as a bovine, wherein the glycosylation pattern may be different from the glycosylation patterns of naturally occurring human lactoferrin obtained from of human milk. DETAILED DESCRIPTION Human lactoferrin can be used for pharmaceutical use (W091 / 13629, incorporated herein by reference), as a nutrient supplement, and for other uses. For such uses, it is often necessary or preferable to employ human lactoferrin which has been purified, either partially or essentially to homogeneity, from unwanted contaminants in milk, especially from other milk proteins (e.g. whey proteins, caseins), milk fat, and other contaminants (for example, lipopolysaccharides of the Gram negative bacterium) present in the milk samples. It has been reported that lactoferrins interact with a wide variety of milk proteins that include IgA, caseins, SC, albumin, lysozyme, lactoglobulin-β and others. The present invention provides purification methods that advantageously support the efficient and rapid purification of lactoferrin, especially human lactoferrin, from milk, such as milk produced by transgenic bovine species harboring a transgene of human lactoferrin which is expressed in the mammary gland One basis of the invention is to find that lactoferrin, especially human lactoferrin, has a surprisingly strong affinity for strong cation exchange resins that can be exploited to purify lactoferrin from milk, especially for purifying human lactoferrin from milk. transgenic bovine species that express a transgene of human lactoferrin. It has also been found that the elevation of the ionic strength of milk or a milk fraction to about 0.2-0.5M NaCl or KCl or an equivalent salt, preferably about 0.35-0.4M NaCl or KCl, more preferably about 0.4M NaCl or KCl, and typically 0.4M NaCl concomitant when contacting the milk or milk fraction with the strong cation exchange resin (s), improves the recovery and resolution of lactoferrin from unwanted contaminants in the milk or milk fraction. The addition of relatively high salt conditions (for example, in a final concentration of approximately 0.4M NaCl) to milk or milk fractions strongly reduces the binding of most contaminating proteins (eg proteins and serum caseins) and lipopolysaccharides ("LPS") to a strong cation exchange resin (e.g., S Sepharose ™ or Mono S ™ Rapid Flow) while allowing efficient binding of lactoferrin (e.g., rhLF) to the strong exchange resin of cation, thus providing a convenient basis for the separation of lactoferrin from contaminating molecular species. It is observed that by raising the ionic strength of the milk or milk fractions to approximately 0.35-0.4M NaCl, the binding of any other bovine-bed protein other than lactoferrin to a resin with strong cation exchange is virtually excluded. The elution profile of bovine serum that has been applied to Mono S under conditions of low ionic strength shows that all bound bovine serum proteins (except lactoferrin) elute at NaCl concentrations of 0.3M NaCl (e.g., bovine lactoperoxidase). ) or lower (other proteins). In addition, it has also been found that strong cation exchange resins and relatively high salt conditions can be used to separate human lactoferrin from bovine lactoferrin in milk or milk fractions. High salt conditions (a concentration of NaCl at least 10 μM higher than physiological milk, usually 0.2M NaCl or more) are used to improve the specific loading of human lactoferrin in strong cation exchange resins. Milk or milk fractions of high ionic strength exhibit a more selective binding of hLF to the selected strong cation exchange resin. In a preferred embodiment, human lactoferrin is expressed and secreted in the milk of a transgenic animal, preferably a bovine species. In those embodiments where the rhLF is expressed and secreted in the milk of transgenic bovine species, the transgenic milk can be used either as obtained or further processed to purify the rhLF. Human lactoferrin obtained by the methods of the invention is preferably obtained by processing a milk fraction, although raw milk (whole milk) can also be used. Preferred milk fractions include milk skimmed milk, skim milk from which particulate matter and / or caseins (for example, whey) have been removed, and other milk fractions containing lactoferrin. Preparation of Milk and Milk Fractions Whole milk is collected from the transgenic non-human animal expressing human lactoferrin. Raw milk (whole milk) can optionally be adjusted to a relatively high ionic strength (eg, 0.3-0.4M NaCl or KCl) by the addition of solid NaCl, KCl, or any other suitable salt, or a solution watery of them. Combinations of monovalent salts (e.g., NaCl and KCl together) may be used, if desired, so that the final concentration of the monovalent cation in milk is about 0.3-0.4M. The whole milk can be contacted with a strong cation exchange resin directly, or it can be processed in a milk fraction that is subsequently contacted with a strong cation exchange resin under relatively high ionic strength conditions (for example, example, 0.3-0.4 NaCl or KCl). If the whole milk is to be contacted with a strong cation exchange resin directly, the ionic strength is adjusted by increasing the salt concentration in the whole milk, typically up to approximately 0.35-0.4M NaCl or KCl, before or concomitant with the contact of the whole milk with the strong cation exchange resin. Optionally, the whole milk can be diluted with an aqueous solution, typically a regulated salt solution, to produce diluted whole milk having a monovalent cation concentration (eg, Na +, K +, or combinations thereof) of about 0.3. -0.4M. Normally a regulated salt solution used as a diluent of whole milk will have a concentration of NaCl (or KCl) of at least about 0.3-0.4M and will be regulated to a pH of 6-8 with an appropriate regulator, - the final concentration of the (the) monovalent cation (s) in the diluted milk is preferably 0.3-0.4M, more preferably 0.4M, and thus the cationic species contributed by a regulator, such as a sodium phosphate buffer, they must be taken into account together with the dissolved salt species (for example, NaCl and KCl). Thus, high ionic strength milk (ie, milk having at least about 0.3-0.4M monovalent cation) can be generated by either adding or dissolving one or more solid salts (e.g., NaCl or KCl). ) in whole milk or when diluting the whole milk in a diluent salt solution whereby the final concentration of the monovalent cation in the diluted milk is approximately 0.3-0.4M. Whole milk of high ionic strength (diluted or undiluted) can be used directly to contact a strong cation exchange resin, or it can be further processed into a milk fraction before coming into contact with a strong resin. cation exchange. Whole milk that did not have the ionic strength increased by the addition of the salt is typically processed in a milk fraction. The ionic strength of the milk fraction is then increased by the addition of one or more salts to increase the concentration of the monovalent cation to about 0.3-0.4M, and the milk fraction of high ionic strength is contacted with a resin of strong cation exchange Without wishing to be bound by any particular theory, dilution of whole milk can also decrease unwanted intermolecular interactions between lactoferrin (eg, rhLF) and contaminating macromolecular species (eg, caseins, LPS from Gram negative bacteria). present in milk. However, significant dilution of whole milk or fractions of milk can result in large volumes that can be processed less efficiently. It has been found that the increase in the ionic strength of milk also substantially decreases the -. unwanted intermolecular interactions between lactoferrin (e.g., rhLF) and contaminating macromolecular species, allowing for easier purification without necessitating the dilution of milk in large volumes, unless desired. Optionally, the addition of a non-interfering detergent (ie, which does not substantially reduce the binding of the hLF to the resin), such as a nonionic surfactant (e.g., Tween-20) in a The concentration of about 0.001-0.2 percent, preferably about 0.01-0.03 percent, can also contribute to the reduction of unwanted intermolecular interactions involving lactoferrin. 15 The processing of whole milk, either directly or after increasing the ionic strength by the addition of salt (for example, NaCl or KCl), in a fraction of milk containing lactoferrin before coming into contact with a strong resin. exchange of cation, can reduce additionally the amount of one or more contaminating species. For example, defatting whole milk may remove a significant proportion of lipid species that may be undesirable and may interfere with purification efficient lactoferrin by chromatography - - strong cation exchange The human lactoferrin obtained by the process of the invention is preferably obtained by the processing of transgenic whey. Whey is a fraction of milk produced by substantially removing all fats and caseins from transgenic milk. A variety of methods (eg, centrifugation) are known to those skilled in the art for removing fats from milk. Similarly, several methods are known to those skilled in the art for removing caseins from milk. For example, acid precipitation or proteolysis of kappa-casein can be used by chymosin to remove casein from milk. Other compatible methods known to those skilled in the art may be used to generate milk fractions containing lactoferrin (e.g., whey). Although salt may be added after removal of caseins from milk, that order of addition may result in the loss of significant amounts of lactoferrin, since caseins are highly phosphorylated in general (ie, negatively charged) and may bind substantial amounts of lactoferrin, presumably through electrostatic interactions, - in this way, the withdrawal of caseins from milk under low conditions salt can result in undesirable removal of substantial amounts of lactoferrin as well. In a preferred embodiment, salt (eg, NaCl or KCl) is added to the whole milk of a transgenic non-human animal expressing human lactoferrin before defatting and removal of caseins; alternatively, the salt may be added after degreasing but before the removal of the caseins. Typically, an aqueous solution containing NaCl and a sodium phosphate buffer is used to dilute the whole milk to form a milk of high ionic strength having a final concentration of 10-50mM sodium phosphate, 0.4M NaCl at approximately a pH of 6.5-7.5. The high ionic strength milk has a final concentration of 20 mM sodium phosphate, 0.4 M NaCl at about pH 7.0. Optionally, a surfactant may be included at a final concentration of about 0.001-0.2 percent by volume, with a typical concentration of about 0.02 percent v / v. Normally, a nonionic surfactant such as Tween-20 is used, other nonionic surfactants may also be suitable. Thus, in this mode, skim milk containing approximately 0.4M NaCl, and optionally containing approximately 0.02 percent of Tween 20, is subsequently produced and used for the removal of the caseins by conventional methods to produce a high ionic strength serum containing lactoferrin. After the previous step, the high ionic strength serum is contacted with a strong cation exchange resin (e.g.

Fluj • o Rá_-pi.do S SefarosaTM, Pharmacia Biotechnology, Piscataway, NJ) under suitable binding conditions whereby the strong cation exchange resin preferentially binds the lactoferrin of the high ionic strength serum to produce a lactoferrin-resin complex. Link of Lactoferrin to Resins of Fuerte Exchange of Cation Human lactoferrin (calculated pl approximately 9.7) does not bind in an essential way to the strong exchange resin of Mono QTM anion at a pH of 4.5 and binds gently to a pH of 7.5 (ie, even at pH values below its pl), which is consistent with the idea that the charge is not evenly distributed over the surface of the human lactoferrin molecule. However, human lactoferrin binds almost completely to strong cation exchange resins, such as Mono S ™ or S Sepharose ™, between about pH 4.5 and 9.5 and at monovalent cation concentrations, equivalent to about 0.5M NaCl or less, preferably 0.45M NaCl or less. This finding is consistent with a hypothesis that human lactoferrin behaves like a molecular dipole, possibly due to the many basic amino acids (Arg and Lys) grouped together in the amino-terminal portion. This encounter provides a basis for the purification of human lactoferrin from milk by reversible adsorption to a strong cation exchange resin under relatively high salt conditions. A multitude of strong cation exchange resins are known to those skilled in the purification technique of the protein. "Strong cation exchange resins" are defined as those that exchange their diffusible cation over a wide pH range, such as those containing sulphite or sulfate groups. Currently, the preferred cation exchange strong resins are, for example, the Mono STM exchange resin and the S Sepharose ™ Fast Flow cation exchange resin (available from Pharmacia / Pharmacia LKB Biotechnology, Piscataway, NJ).

The STM Monkey Monoblock exchange terminals and the S Sepharose ™ Fast Flow currently use the S-ligand, although the SP ligand is also suitable and can be replaced. Resins with similarly charged groups that provide strong cation exchange will also be useful for the purposes of the invention. A variety of methods can be used to contact milk or milk fraction, including transgenic bovine serum containing human lactoferrin with a strong cation exchange resin. For example, the strong cation exchange resin can be formed in a column and the serum containing lactoferrin can be passed through the column. After adsorption of the lactoferrin to the column resin, the column is rinsed and the human lactoferrin is subsequently desorbed by elution at a sufficient ionic strength to elute the human lactoferrin, preferably at a sufficient ionic strength to elute the human lactoferrin in a manner efficient but without substantially eluting bovine lactoferrin, if present. Alternatively, the lactoferrin-containing serum can be contacted with a strong cation exchange resin in the form of a bed, generally under conditions of relatively high ionic strength (eg, 0.35-0.4M NaCl, pH 7.0). ). In this case, the lactoferrin-containing serum is contacted with the strong cation exchange resin in a bed and stirred for a period of time suitable at a suitable temperature (eg, 4.30 degrees Celsius); Such contact can be carried out by mixing the resin beads directly in the serum containing lactoferrin. Subsequently, the strong cation exchange resin with the adsorbed lactoferrin is separated by, for example, centrifugation, spontaneous sedimentation, or by forming in a column, and the mobile phase (ie, lactoferrin-drained serum) is substantially removed. The separated lactoferrin-resin complexes can be rinsed to remove the lactoferrin-free species bound to the resin or remaining unbound, if desired, with a rinse regulator having a sufficiently low ionic strength (e.g., the concentration of the monovalent cation is less than about 0.45M) in order to avoid substantial elution of lactoferrin from the resin, but preferably high enough to elute macromolecules without lactoferrin that may or may not bind to the resin. Typically, a rinse regulator may comprise between about 0.01-0.45M NaCl, 5-50mM sodium phosphate buffer at about a pH of 7.5, although other rinsing solutions may be used. For example, a rinse regulator containing 0.3M of NaCl, lOmM of sodium phosphate at a pH of 7.5. Optionally, a detergent such as a nonionic surfactant (e.g., Tween-20) can be included in a rinse regulator, typically at 0.01-0.1 percent by volume. The separated lactoferrin-resin complexes can be rinsed in a single rinse step, in multiple rinse stages, or they can remain un-rinsed. In general, at least one volume of rinsing regulator per unit volume of the resin is used to rinse the separated lactoferrin-resin complexes, often higher rinsing volumes are used, especially if the rinsing regulator has a low ionic strength ( for example, less than 0.2M NaCl), if the rinse is carried out. The rinsing serves to remove the lactoferrin-free macromolecules from the resin either as a column or as a bed. The lactoferrin is selectively desorbed from the lactoferrin-resin complexes separated by elution with a solution of appropriate ionic strength. In general, an elution buffer will comprise a monovalent salt (eg, NaCl or KCl) in a concentration of at least about 0.3-0.4M, typically 0.45-0.5M or greater, and preferably also contains a suitable buffer, such as sodium phosphate (eg, 5-50mM) at a pH of about 7.5. A possible variety of compositions and salt concentrations of the eluting solution and can vary with the purification scale, so that the calibration or standardization of the performance of the column or bed of resin can be carried out by those skilled in the art using methods of calibration standards. Alternatively, human lactoferrin can be selectively desorbed from the lactoferrin-resin complexes packed in a column by elution with a gradient of NaCl or KCl or a step gradient as the mobile phase, typically occurring the desorption of human lactoferrin to approximately 0.30- 0.75M of Nací, depending on the scale of purification and other performance factors of the column. Calibration of the performance of the column and determination of the elution profile can be easily determined by those skilled in the art for each modality. The human lactoferrin-containing fraction (s) can be identified by any convenient assay for human lactoferrin, including but not limited to: an immunoassay using the antibody that specifically binds human lactoferrin (Nuijens et al. (1992) J) Lab. Clin. Med. 119: 159) or other assays to detect and quantify lactoferrin. In a further embodiment, lactoferrin is Remove from the lactoferrin-resin complexes by dialysis. Dialysis is a widely practiced technique known to those skilled in the art. The dialysis membrane is selected in such a way that the protein, for example human lactoferrin, passes unimpeded from one side of the membrane to another. In the present case, there is a strong cation exchange resin on the side of the membrane opposite the side that contacts the milk or fraction of transgenic milk containing lactoferrin. Lactoferrins, which include human and bovine lactoferrin, are divided from the milk or milk fraction through the dialysis membrane to the side containing the strong cation exchange resin, where they bind to the resin of strong cation exchange . The pore size (molecular weight cut) of the selected dialysis membrane prevents the lactoferrin-resin complexes from passing through the membrane into the milk or milk fraction. In this embodiment, the dialysis membrane is selected such that only unbound lactoferrin, and not lactoferrin bound to the strong cation exchange resin, passes through the membrane. Typically, the milk or milk fraction is adjusted to an ionic strength of about 0.35-0.4M monovalent salt (NaCl or KCl) and is generally regulated to a pH of about 7.5, normally, with an appropriate regulator (eg, sodium phosphate, potassium phosphate) before or concomitant with contact of the milk or milk fraction with the dialysis membrane that separates the resin from strong cation exchange from the milk or fraction milk. Optionally, a detergent, such as a nonionic surfactant (e.g., Tween-20), may be included in a concentration of about 0.001 to 0.2 percent v / v, preferably about 0.02 percent v / v, if present. After a period of adequate dialysis to allow the partition of the lactoferrin through the membrane to bind to the strong cation exchange resin, the milk or milk fraction thus emptied of the lactoferrin is removed and the lactoferrin bound to the resin of strong cation exchange is eluted, either directly or through a dialysis membrane, by contacting the lactoferrin-resin complexes with a high ionic strength regulator (eg, 0.45-0.7M NaCl or KCl, 5). -50mM of sodium phosphate or potassium phosphate at a pH of 7.5). A variety of alternative aqueous solutions can be used to elute the lactoferrin from the strong cation exchange resin and will be apparent to those skilled in the art. It can also be used by those skilled in the art to practice invention, the substitution of other salts (for example, LiCl), including the salts of divalent cations. The lactoferrin eluted from the lactoferrin-resin complexes can be subjected to an additional round of cation exchange chromatography and / or lectin affinity chromatography (eg, Con A) to further reduce the human lactoferrin of bovine lactoferrin, if it occurs. In addition, the rechromatography can be used to further purify the intact human lactoferrin from the degradation products (proteolysates). Preferably, the intact human lactoferrin is recovered in substantially pure form. After eluting the lactoferrin from the lactoferrin-resin complexes bound with a salt solution of high ionic strength, the eluted lactoferrin can be treated to clarify and concentrate. Preferably, the recovered lactoferrin is dialyzed to remove the salts, ultrafiltered, and optional ly lyophilized in order to concentrate the lactoferrin. Suitable storage and / or reconstitution components (eg, regulators, salts, preservatives, antibiotics, ferric ions) can be added to the purified lactoferrin.

Purification of Lactoferrin from Milk by Discontinuous Extraction It is believed that the discontinuous extraction of lactoferrin from milk, through the use of a strong cation exchange resin, is a preferred purification strategy for large-scale industrial applications. In discontinuous extraction, it is usually * > • It is preferable that the salt be added to the milk or fraction of Milk in order to produce a high ionic strength milk solution (eg, 0.4M NaCl) prior to, or concomitant with, the contact of the strong cation exchange resin with the milk solution. It is believed that salt is added to increase the resistance sufficient to (1) reduce potential intermolecular electrostatic interactions between contaminants (eg, caseins and LPS) and lactoferrin, (2) reduce the binding of lactoferrin-free macromolecules to the strong exchange resin. cation, that is, ensure the selectivity of the lactoferrin bond in the case that a non-limiting amount of a strong cation exchange resin is added (protein binding capacity of more than the amount of lactoferrin species object) to the milk or fraction of milk, (3) induce the aggregation of certain species without lactoferrin that can be removed later before a column is prepared, and (4) avoid aggregation of materials (possibly including lactoferrin) that can be retained in the resin under low salt conditions and captured in the chromatography column when apply a high ionic strength rinse or a salt gradient, thus preventing the performance of the column and allowing partial disaggregation and elution of the aggregate to contaminate the eluted lactoferrin. However, the ionic strength should not be excessively high so that lactoferrin is not efficiently bound by the strong cation exchange resin. For example, an ionic strength of about 0.35-0.45M NaCl is generally preferred, typically 0.4M NaCl being used. In addition, when lactoferrin is extracted from milk fractions such as whey (for example, prepared by acid precipitation, chymosin treatment, or ultracentrifugation), the recovery of lactoferrin is increased if the salt has been added to increase the ionic strength before the casein withdrawal. This effect is presumably due to the retention of lactoferrin by the electrostatic interaction with the caseins and can be overcome by increasing the ionic strength before the withdrawal of the casein. As an example and not as a limitation, the milk of a transgenic non-human animal expressing rhLF can be processed according to the following protocol to substantially purify human lactoferrin from milk. First, a concentrated solution of 5M NaCl or solid NaCl is added to the milk to produce a final concentration of 0.35-0.45M NaCl, usually about 0.4M NaCl. Optionally, a non-ionic surfactant (Tween-20) of up to 0.02 percent (v / v) final concentration is added. Optionally, sodium phosphate is added to a final concentration of approximately 20 mM with an expected pH of approximately 7. 5, for example by adding NaH2P04 »H20 and Na2HP04 * 2H20 to a final concentration of 3.2mM and 16.8mM, respectively; The final pH of the milk solution does not need to be exactly 7.5, and will often be about 6.5-7.0. It is believed that the addition of sodium phosphate (or other regulatory agent) can be omitted, since hLF binds efficiently to strong cation exchange resins (e.g., Mono-S ™ or S Sepharose ™) at values of pH between 4.5 and 9.5. The milk fat is removed by centrifugation at 1600 x g for 10 minutes in 500 ml polyialomer tubes in a Beckman JA-10 rotor to produce a skim milk high ionic strength. Alternatively, spontaneous sedimentation followed by physical removal of the fat layer can be used. Alternatively, the milk fat can be removed by centrifugation after incubation in batch form of the milk processed with a strong cation exchange resin. Typically, a strong cation exchange resin (eg, S Sepharose, Pharmacia LKB, Piscataway, NJ) is typically equilibrated with a high ionic strength regulator (0.4M NaCl, 20mM sodium phosphate, a pH of 7.5, optionally including 0.02 percent Tween-20) and dissolved in the processed milk. Approximately 1 ml of balanced cation exchange strong resin beads, packed, balanced with a high ionic strength regulator for 5-20 mg lactoferrin in the processed milk sample (which can be determined by a lactoferrin assay) is added and stirring during a suitable mixing period (e.g., about 1 hour or more, preferably overnight) to bind the lactoferrin to the resin beads. Resin pearls (e.g., Fast-S ™) are then pelletized by centrifugation at approximately 1600 x g for 5 minutes and the supernatant is removed. Alternatively, the beads can be made pellets by spontaneous sedimentation after which the whole or skimmed milk of high ionic strength emptied of lactoferrin is removed. The pelletized resin beads are rinsed three times with about one volume of high ionic strength regulator, and resin beads with a volume of rinse regulator are then spilled into a column. A substantially purified preparation of lactoferrin is eluted from the column with a gradient (i.e., 1.25 times the volume of the column) of 0.4-1.0M NaCl in 20mM sodium phosphate at a pH of 7.5. The excess salt present in the recovered lactoferrin can be removed by dialysis against saline or phosphate buffered saline (dialysis against distilled water tends to cause precipitation of lactoferrin). Human lactoferrin can be separated from endogenous lactoferrin (non-human, - for example, bovine) by eluting the column with a salt gradient or stepwise elution, the native human lactoferrin (e.g., rhLF) being eluted from the column at a resistance lower or higher ionic than non-human lactoferrin (for example bovine or porcine, respectively) at a pH of 9.5 or lower. For example, when a 20mM sodium phosphate buffer is used at a pH of 7.5, the rhLF is typically eluted at a concentration of NaCl that is approximately 50-100mM (typically 70mM) lower than the NaCl concentration at which efficient elution of bLF occurs. If further purification of the bLF rhLF is desired, the recovered fractions containing substantial amounts of rhLF can be rechromatographed on a strong cation exchange resin and / or Con A column and eluted with a salt gradient or step elution to remove in addition, the hLF of the bLF in the eluted fractions. Formulations of Lactoferrin Lactoferrin produced by the methods of the invention is substantially purified. That is to say, lactoferrin is substantially free from contamination with other milk proteins and molecular contaminants present in milk, including bacterial lipopolysaccharides. Human lactoferrin produced by the methods of the invention is suitable for formulation in pharmaceutical or food supplements comprising lactoferrin, typically, comprising from at least about 1 milligram to several grams or more of lactoferrin per dose. In view of the antibacterial and anti-inflammatory properties of human lactoferrin, a variety of uses for purified human lactoferrin are possible. For example, pharmaceutical and / or nutritional antibacterial supplements comprising lactoferrin, especially human lactoferrin, typically in conjunction with other agents, to a patient for therapy or prophylaxis of local infection, infection (bacterial) on a large scale, blood-borne infection (sepsis) as well as inflammation resulting from infection or non-infectious inflammatory diseases (for example, chronic inflammatory disease of the colon or ileum). Such pharmaceutical compositions are prepared containing lactoferrin, preferably human lactoferrin, in combination with a physiologically compatible carrier, typically for administration as an aqueous solution (eg, in physiological saline) via intravenous, intramuscular, subcutaneous, topical routes of administration. fattening, washing, or other suitable. Similarly, pharmaceutical and nutritional preparations containing substantially purified lactoferrin can be used, typically in conjunction with other pharmaceutical or nutritional agents, to treat large-scale bacterial infections. For example, pharmaceutical preparations containing human lactoferrin can be used to treat bacterial infections housed in the blood, either alone or in conjunction with another pharmaceutical agent, or used to prepare or treat organ transplant recipients or other immunosurbed individuals (eg, patients with AIDS) against the effects of infections. Additionally, substantially purified human lactoferrin can be used in the formulation of nutritional supplements. For example, for human use, purified human lactoferrin can be included in infant formula or can be used as a dietary supplement for adults. As a result of the iron binding properties of lactoferrin, nutritional preparations useful for treating iron deficiencies (eg, iron deficiency anemia of premature infants) can be formulated with substantially purified lactoferrin. Quality Control Assays for Lactoferrin The quality control of the purified hLF must include at least one, preferably more, optionally all of the following procedures: (1) Reduced and unreduced SDS-PAGE, with samples loaded without boiling before and after boil, (2) spectroscopic analysis such as absorption measurement at 280 and 465 nm, (3) analysis of radioimmunoassay of hLF and bLF, (4) chromatography of strong ion exchange • • • • (eg Mono STM ), and (5) sequencing of the N-terminal protein. The following examples are offered as an illustration and not as a limit to the invention.

EXPERIMENTAL EXAMPLES Effect of Ionic Resistance in the Recovery of Lactoferrin We have found that the addition of salt to transgenic mouse and bovine milk before the caseins are removed significantly increases the production of transgenic hLF and bLF, respectively, after purification of the serum fraction. The data presented in Table 1 provide the background for these differential recoveries. Table 1 bLF was determined by competitive inhibition of RIA in the whole bovine milk samples (n = 10) as well as in the whey and casein fractions prepared after centrifugation for 30 minutes at 10,000 g. Acid precipitation was carried out by adjusting the whole milk to a pH of 4.6, followed by incubation of the milk for 30 minutes at 40 ° C. The results (mean + SD) are expressed as a percentage of the total bLF found in the respective fractions. The concentration of -bLF in the casein pellets was on average 4.0 times higher than in the serum fraction. The concentration of -bLF in the casein pellets was on average 8.0 times higher than in the serum fraction. Effect of the Ionic Resistance and the Detergent in the Human Lactoferrin Link The effects of the salt concentration and the presence of a nonionic surfactant were determined.

(Tween-20) or cationic surfactant (Polybrene) in the binding of human lactoferrin (hLF) labeled with 125 I in various ligands immobilized in Sepharose. Sepharose was suspended in 10mM of sodium phosphate, 0.15M NaCl, at a pH of 7.4. The following ligands were immobilized on the Sepharose and used for the binding determination for hLF: R595, an LPS of S. Minnesota (KDO +, no antigen of 0, rough), - J5 an LPS of E. Coli (KD0 +, without antigen of 0, rough), - heparin, a polyanion known to bind to lactoferrin; HSA, human serum albumin; and glycine. The labeled 125I hLF was contacted with the Sepharose ligand resins and exposed to various combinations of NaCl concentration and detergent. Table 2 shows the percentage of 125 I-hLF (ie, radioactivity) retained in the various Sepharose ligand resins. Table 2 Link Percentage of --I-HLF to Various Sepharose Table 2 indicates that the ionic strength of more than about 0.25M NaCl and the 0.2% Tween-20 detergent concentration substantially reduces the binding of hLF to the various ligand species, including bacterial LPS. This experiment indicates that the interaction between LPS and hLF is electrostatic in nature. Studies were conducted to determine the binding of hLF to LPS types from a broad range of I "Variety (ie, more than 50 types) of clinically relevant Gram-negative bacteria." Lactoferrin reacted with varying affinities to each type of LPS evaluated.Lactoferrin appeared to interact electrostatically with the lipid A residue of the LPS, to a site Identical or in close proximity to the interaction site of LPS with B Polymyxin, an antibiotic known to neutralize the toxic effects of LPS Elution Pattern of Lactoferrin from Ion Exchange Resins Fifty micrograms of protein (hLF Sigma 1 - see legend of Table 5 - and bLF, Sigma, St. Louis, MO) in various different pH regulated solutions were applied to HR5 Pharmacia / 5 columns containing 1 ml of M "and M resin Mono S (strong cation exchanger) or Mono Q (strong anion exchanger) balanced in appropriate regulators using the FPLC system (Pharmacia, Piscataway, NJ) for the determination of elution profiles with NaCl in various regulators of various pH (sodium succinate, sodium acetate, sodium phosphate, Hepes, Tris, ethanolamine, N-methylpiperidine). After rinsing each column with 2 ml of the regulator, a linear salt gradient was applied from 0-1. OM NaCl in 30 ml of regulator at a flow rate of 1 ml / min. HE monitored the peaks by measuring absorption to 280nm. The elution of hLF and bLF in various regulatory systems is shown in Table 3. Table 3 Elution Pattern of LF Preparations in Mono S ™ and Mono Q ™ Columns a - about 21% of the applied hLF was not bound to the column, b - about 14% of the applied hLF was not bound to the column. Table 3 shows that hLF binds almost completely to Mono S TM between the pH of 4.5 and 9.5, indicating that the basic amino acid residues are grouped in the hLF. However, the N-terminus of the hLF contains residues basic amino acids grouped and thus the hLF appears as a macromolecular dipole. The hLF is eluted in three peaks, in contrast to the theoretical data. The reduced and unreduced SDS-PAGE analysis of the hLF I, II and III peaks (see Tables 2 and 4) did not reveal any unfolding of the hLF. All peaks contained the same proportion of Fe-hLF (SDS-PAGE not reduced, without boiling). The amount of hLF detected by the RIA corresponds to the chromatogram. TM The hLF does not bind Mono Q at a pH of 4.5 and binds smoothly to a pH of 7.5 (the pH value below its pl). This correlates with the idea that the charge is not evenly distributed over the hLF molecule. Based on these discoveries, we selected a strong cation exchange resin at an approximately neutral pH for the purification of hLF. Discontinuous Purification of Lactoferrin from Milk Lactoferrins were purified from human milk, bovine milk and transgenic mouse milk expressing rhLF in their milk by the discontinuous extraction method. Solid NaCl was added to a final concentration of 0.4M and Tween-20 was added at a final concentration of 0.02% (v / v). The sodium phosphate buffer (pH 7.5) was added to 20mM of the final concentration, but the final pH was not adjusted to 7.5. Milk fat was removed by centrifugation for 10 minutes at 1600 x g in 500 ml polyalomer tubes in a Beckman JA-10 rotor. The S Sepharose TM Fast Flow packaged, equilibrated with a start buffer (0.4M NaCl, 20mM sodium phosphate, pH 7.5, 0.02% Tween-20) was added to the processed milk in a ratio of approximately 1 ml of resin pearls packed with 5-10 mg lactoferrin in the processed milk. The mixture was stirred for 20 hours, and the resin beads were separated by centrifugation at 1600 x g for 5 minutes. The supernatant was removed and the beads were rinsed three times with one volume of the start buffer. The resin was then poured into a column and rinsed with a volume of 20 mM sodium phosphate, 0.4 M NaCl, at a pH of 7.5. Lactoferrin was eluted from the column with a gradient of 0.4-l.OM NaCl at 1.25 (times the column) the volume of 20mM sodium phosphate, a pH of 7.5, with a flow rate of 10 ml / min. The results of the lactoferrin recovered by this method are shown in Table 4, which tabulates the quality of the purified lactoferrin samples as determined by gel electrophoresis, spectroscopy and chromatographic performance on the Mono S ™ resin.

Table 4 Evaluation of Purified LF Preparations 1. Purity was determined by applying the samples of at least 10 μg of protein to the gel electrophoresis (SDS-PAGE) thick film of SDS-polyacrylamide (7% w / v). The protein bands were visualized by staining with Coomassie Brilliant Blue. The percentage of iron saturation of lactoferrin was determined by SDS-PAGE not reduced, without boiling. The cleavage of lactoferrin was determined by the reduced SDS-PAGE. 2. Purity was determined by absorbance spectrum analysis; for the calculations, we used an A, of 0.1%, 1 cm at 280 nm of 1.1 for the unsaturated LF (apo), an A, of 0.1%, 1 cm at 280 nm of 1.4 for the saturated LF (Fe), and an A, of 0.1%, 1 cm at 465 nm of 0.056 for the hLF of Fe. 3. The purity was determined by applying 1 mg of protein on a Mono S ™ column (1 ml) with full-scale sensitivity of 0.01. Nativity (see below) of hLF was analyzed by applying 50 μg of protein in the column and expressed as the% total hLF eluted at the peak III position (0.7 NaCl). The elution program was a linear salt gradient (0-1M NaCl) in 30 ml of 20 mM sodium phosphate, at a pH of 7.5 - The bLF was eluted as a single peak with a small protrusion at approximately 0.8 M NaCl - rhLF represents transgenic recombinant hLF purified from transgenic mouse milk. The dose response curves of purified natural hLF and transgenic rhLF as determined in a sandwich-type RIA for hLF were parallel to the standard curves of hLF derived from commercially available purified human milk. Sequential analysis of the amino terminal protein revealed that the signal sequences of the purified natural hLF and transgenic rhLF were correctly and completely removed and showed no degradation of the N-terminus. The dose response curves of purified bLF as determined in a RIA of competitive inhibition specific for bLF were parallel to the standard curves of bLF derived from commercially available bovine milk. Evaluation of Purified hLF Preparations Using Mono S ~ TM Chromatography During the course of purification studies with transgenic rhLF from mouse milk (0-1M linear gradient of NaCl in 20mM sodium phosphate "or at a pH of 7.5 in a Mono STM column), we observed that at least about 95% of transgenic rhLF was eluted at 0.7M NaCl, while commercially available hLF preparations (Sigma and Calbiochem) purified from human milk eluted as three peaks at approximately 0.5, 0.6, and 0.7M NaCl (denoted to hLF peaks I, II and III, see also Tables 3 and 5). The hLF that was purified from fresh human milk was always eluted at approximately 0.7M NaCl. The bLF was eluted to approximately 0.8M NaCl. Table 5 shows the elution of the patterns of various preparations of lactoferrin and others in a column of Table 5 Elution Pattern of Various LFs and Other Preparations in a Mono S TM Column 50 μg of protein in 20 M sodium phosphate was applied, at a pH of 7.5 (regulator A) to a Mono S ™ column (Pharmacia HR5 / 5 containing 1 ml of resin) using the FPLC system (Pharmacia ). After rinsing the column with 5 ml of buffer A, a linear salt gradient was applied from 0-1.0M NaCl in 30 ml of buffer A at a flow rate of 1 ml / min. The peaks were monitored by measuring absorption at 280 nm with a full-scale sensitivity of 0.01 using a flow cell of 0.2 cm. The following abbreviations: hLF Sigma 1, hLF "native" from Sigma derived from purified human milk, - hLF Sigma 2, Sigma 1 repeatedly frozen and dissolved; hLF Sigma 3, a different part of the "native" hLF of Sigma; Calbiochem hLF, the hLF of Calbiochem derived from human milk, - hLF GPE 1, 2, and 3, the hLF preparations with iron saturation levels of approximately 3% purified by strong cation exchange chromatography of human milk sample fresh from three donors with a total iron saturation of 3%; hLF GPE 4, the hLF purified from a human milk sample that has been stored for a week at 4 ° C; FehLF Sigma 1 and 2, different parts of hLF derived from purified human milk that is completely saturated with iron by Sigma; Arg-mod. hLF GPE, hLF derived from purified human milk that has had chemically modified Arg residues; FehLF GPE, the hLF derived from purified human milk saturated with Fe, - Deglyc. hLF GPE, hLF derived from purified human milk that was deglycosylated completely with N-glycosidase, - Neura. hLF GPE, hLF derived from purified human milk that has had sialic acid residues removed with neuraminidase; Tryp. hLF GPE 1, 2, and 3, the hLF derived from purified human milk that was incubated with trypsin (the molar ratio of hLF: trypsin was 7.5: 1) for 1 minute, 3 hours and 24 hours, respectively, followed by the addition of soy bean trypsin inhibitor, - Trans. hLF GPE 1 and 2, purified hLF from mouse milk harboring cDNA (codes for bovine casein signal sequence aSl bovine fused with mature hLF) and transgenic genomic hLF constructs expressing rhLF at 0.2 and 2.0 mg / ml, respectively; 293 rhLF, recombinant hLF expressed in tissue culture (293 cells); 293 Unglyc. rhLF, undivided, non-glycosylated, purified rhLF expressed by 293 cells in the presence of tunicamycin, - Sigma bLF, Sigma bLF derived from purified bovine milk; bLF GPE, bLF purified from fresh bovine milk by strong cation exchange chromatography; Arg-mod. bLF GPE, purified bLF that has had chemically modified Arg residues; mLF GPE, murine lactoferrin purified from fresh mouse milk by strong cation exchange chromatography, - mDF GPE, murine domferrin purified by high cation exchange chromatography in mouse milk (mDF is a protein of 80 kD belonging to the transferrin family), - purified mTF Sigma, (sero-) transferrin from mouse acquired from Sigma; hTF Sigma, purified human serotransferrin acquired from Sigma; pLF GPE, porcine lactoferrin purified from porcine milk by strong cation exchange chromatography; Goat serum and goat serum, serum fractions prepared from mutton and goat milk respectively. In theory, Makino et al. (1992) J. Cromato. 579: 346 reports that the three peaks of hLF that were eluted from a Mono S column at 0.88, 0.97, and 1.05M NaCl represent diferric, monoferric, and apolactoferrin, respectively. However, our results of saturation studies of hLF with iron indicate that the two ferric atoms are incorporated in a coordinated manner in each hLF molecule, so that essentially all the hLF in the milk is apolactoferrin or diferric. Native hLF (hLF purified from fresh human milk, -only 3% saturated with iron by the measurement of absorbency) and Fe-hLF (completely saturated with iron as determined by the absorbance measurement and the SDS-PAGE not reduced, unboiled) is eluted in exactly (within experimental resolution limits) the same position (approximately 0.7M NaCl) from a Mono S TM column at a pH of 7.5. The native hLF, completely deglycosylated (with N-glycosidase), and treated with neuraminidase is eluted in exactly (within the limits of experimental resolution) the same position (approximately 0.7M NaCl) from a Mono STM column to a pH of 7.5.

The relative amount of hLF I and II peaks increased after prolonged dialysis (4 days) of transgenic mouse serum. In addition, the hLF II and I peaks appeared after limited tryptic proteolysis of the native hLF (peak III) before the degradation of hLF could be observed by the reduced or unreduced SDS-PAGE. Based on these observations, peaks II and I in the hLF preparations could be generated by limited proteolysis of peak III (native hLF), such as with the separation of the serine protease in arginine. In accordance with this idea, the results of the analysis of the amino-terminal protein sequence of the hLF peaks I, II, and III present in the commercially available purified hLF were obtained (see Tables 6 and 7). N-terminal proteolysis of hLF may be important since the biological activities of the truncated hLF variants may be different from those of native hLF, such as the binding of hLF to cellular receptors, the rate of evacuation in vivo in the circulation , the ability to inhibit the endocytosis of the remaining chylomicrons, and / or antibacterial properties. Tables 6 and 7 show the N-terminal sequence analysis of some of the proteins in Table 5.

TABLE 6 Sequential analysis of N-terminal protein of lactoferrin and some species related to lactoferrin (amino acids 1-25) 1 5 10 hLF GPE1 qlv-arg-arq-arq-arq-ser-val-qln-trp-xxx- ala-val-ser-hLF Calbi .picoIII lv-arq-arq-arq-arg-ser-val-qln-hLF Calbi. picoll arq-arq-arq-ser-val-qln-trp-xxx-ala-val-ser-hLF Calbi. picol arg-arq-ser-val-gln-trp-xxx-ala-val-ser-hLF GPE4 picoIII qlv-arq-arq-arq-arq-ser-val-qln-trp- Tr.hLF GPEipicoin qly-arq- arq-arq-arq-ser-val-qln-trp-xxx-ala-val -ser-hLF cdn GPE qlv-arq-arq-arq-arq-ser-val-gln-trp-cvs-ala-val-ser - Tr.hLF GPE2 qlv-arq-arq-arq-arq-ser-val-qln-trp-xxx-wing-val-ser-mLF GPE lvs -ala- thr-thr- val -arg- tr -xxx- wing - val-ser -mLF cdn Teng lvs - ala-thr-thr-val -arg-rp -cvs- ala-val-ser -mDF GPE lvs-ala-val-arq-val-qln-trp-xxx-ala- val-ser-mTF Sigma val -pro -asp -lys -thr -val -lvs -trp -xxx- ala-val -xxx -hTF val -pro- asp -lvs -thr- val -arg-trp- cvs -ala -val- ser -bLF cdn Pierce ala-pro- argys -asn-val-arg-trp -cvs- thr -ile- ser -oLF ala-pro- arqys -asn- val -arg-trp -cvs -ala -i le -ser-pLF ala-provs -lys-gly-val -arq- trp- cvs -val -i le -ser- The proteins given in italics are derived from the theory. The underlined amino acids represent the basic amino acid residues.

TABLE 6 (Cont / d.) 20 25 hLF GPE1 qln-pro-glu-ala-thr-lvs-xxx-phe-qln-trp-gln-arq- hLF Calbi. icol gln- Tr.hLF GPElpicoIII gln-pro-hLF cDNA GPE gln-pro-glu-ala thr-lvs-cvs-phe-gln-trp-gln-arq- Tr.hLF GPE2 gln-pro-glu-ala xxx- lvs-xxx-phe-gln-mLF GPE asn-ser-glu-glu • qlu-lvs-xxx-leu-arg-trp-qln-mLF cDNA Teng asn-ser-glu-glu qlu-lys-cys-leu arg-trp-qln-asn-mDF GPE asn-glu-glu-mTF Sigma glu-his-xxx-asn -ile-lvs-hTF glu-his-glu-ala -thr-lvs-cys-gln-ser-phe -arq-asp-bLF cDNA Pierce gln-pro-glu-trp • phe-lvs-cys-arg-arg-trp-gln-trp-oLF pro-pro-glu-gly-ser-arg-cys-tyr-gln -trp-gln-lys-pLF thr-ala-glu-tyr -ser-lvs-cys-arg-qln-trp-qln-ser- TABLE 7 Sequential analysis of N-terminal protein of lactoferrin and some species related to lactoferrin (amino acids 26-50) 26 30 35 hLF cDNA GPE asn-met-arq-lvs-val-arq-qlv-pro-pro-val -ser-cys-mLF cdn Teng lu-met-arq-lvs-val-gly-qly-pro-pro-leu-ser-cys-hTF his-met-lvs-ser-val-ile-phe-ser-asp -qly-phe-ser-bLF cdn Pierce arg-met-lvs-lvs-leu-glv-ala-phe-ser-ile-thr-cvs-oLF lvs-met-arg-arg-met-pLF lys- ile- arg-arg-thr-asn-pro- ile-phe-cvs-ile-arg- TABLE 7 (Cont / d.) 40 45 50 hLF cDNA GPE ilevs-arg-asp-ser-phe-ile-gln-cys-ile-qln-ala-ile-mLF cDNA Teng val-lvs-lvs-ser-ser-thr-arg-gln -cvs-ile-gln-ala-ile-hTF val-ala-cvs-val-lvs-lvs-ala-ser-tyr-leu-asp-cys-ile-bLF cdn Pierce val-arg-arg-ala-phe -ala-leu-glu-cys-ile-arg-ala-ile-pLF arg-ala-ser-pro-thr-asp-cvs-ile-arq-ala-ile- On the basis of the elution patterns of Table 5, the sequential data of the N-terminal protein of Tables 6 and 7 as well as those of the transferrins and lactoferrins published in the theory, we conclude that the amino-terminal sequences of the transferrins / lactoferrins determine the binding characteristics of these molecules to the resins of strong exchange of cation and that the native hLF (peak III) can be purified from degradation products (peaks I and II) by strong cation exchange chromatography. In this way, strong cation exchange chromatography can be used for quality control assessment of hLF by separating and quantitatively detecting the amounts of native hLF and degradation products (peak material I and II), and preparative purification of native hLF from such degradation products, such as for the pharmaceutical formulation of homogenous native hLF or purified peak I or peak II material, if desired. On the basis of the differential elution patterns of the human and non-human lactoferrin species as presented in Table 5 as well as from experiments with mixtures of purified hLF and bLF, and bovine milk omitted with hLF in the Mono S TM (see below), we also conclude that the separation of the rhLF produced transgenically from Non-human lactoferrin (e.g., bLF) can be carried out with strong cation exchange chromatography by the use of a salt gradient or step elution with regulators of increasing ionic strength (salt concentration). It should be noted that the transgenic rhLF is produced in the milk of the transgenic mouse eluted at the same ionic strength as the hLF derived from human milk. In addition, all the proteins from the sheep and goat serum fractions that have been bound to the Mono S TM were elucidated at NaCl concentrations lower than 0.6M NaCl (Table 5). This indicates that the transgenic rhLF can be reduced from the goat and goat lactoferrin species by strong cation exchange chromatography. Chromatographic separation of hLF and bLF Table 8 shows the elution patterns of hLF and bLF in Mono STM with linear salt gradients that demonstrate the different salt resistances in which hLF and bLF are eluted.

TABLE 8 Effect of pH on Elution Pattern of HLF and BLF on Mono S TM Fifty μg of protein (Sigma 3 from hLF and / or Sigma from bLF) were applied in the regulators of different pH (buffered A) to the column (Pharmacia HR5 / 5 containing 1 ml of balanced resin beads) that was used the FPLC system of Pharmacia. After rinsing the column with 5 ml of buffer A, a linear salt gradient from 0-1.0M NaCl in 30 ml of buffer A was applied at a flow rate of 1 ml / minute. The peaks were monitored by absorption measurement at 280 nm.

Since the difference in elution patterns was relatively insensitive to pH at the pH values examined, the optimization of the separation of hLF and bLF in the Mono S TM was determined at an almost> pH. fi • si • oló _. * gi > co (pH of 7.5). As shown in figure 1, they were applied 100 μg of Sigma 3 from hLF (panel A) or Sigma from bLF (panel B) in regulator A (20 mM sodium phosphate, pH 7.5) to the Mono S ™ column (Pharmacia HR5 / 5 containing 1 ml of balanced resin beads) using the Pharmacia FPLC system. After rinsing the column with 5 ml of buffer A, a linear salt gradient was applied from 0-1. NaCl OM in 30 ml of regulator A at a flow rate of 1 ml / minute. The peaks were monitored by absorption measurement at 280 nm (total scale of 0.02). Figure 2 shows the differential elution pattern of hLF and bLF in Mono S with a stepped elution program (sequential increments of salt concentration up to 0.6 and l.OM NaCl). 100 μg of Sigma 3 of hLF (panel A) or Sigma of bLF (panel B) in regulator A were applied to the Mono S ™ column (Pharmacia HR5 / 5 containing 1 ml of balanced resin beads) using the Pharmacia FPLC system.

After rinsing the column with 5 ml of buffer A, the salt concentration was increased stepwise from 0M to 0.6M NaCl in buffer A, at a pH of 7. 5 at a flow rate of 1 ml / minute. After 10 minutes, another increment was applied stepwise from 0.6M to l.OM NaCl in regulator A at 1 ml / minute. The peaks were monitored by absorption measurement at 280 nm. Figure 3 shows the substantial purification of hLF and bLF by resolution of a mixture of purified proteins on a Mono STM column with either a linear salt gradient or stepped elution. 100 μg of hLF and 100 μg of bLF were applied to the column in regulator A and eluted by the linear NaCl gradient (panel A) or by step elution (panel B) as described above. The main peak of hLF is eluted at 0.67M NaCl and the main peak of bLF is eluted at 0.75M NaCl under the conditions used for the linear gradient elution (Figure 3, panel A). With the stepped elution program, the main peak of hLF is eluted in a stage regulator of 0.6M NaCl and the main peak of bLF is eluted in the l.OM NaCl stage regulator (figure 3, panel B) . Figure 4 shows the specific radioimmunoassays for quantifying hLF and bLF in the elution fractions of the elutions in a gradient fashion (panel A) and in a staggered fashion (panel B) under figure 3. The purity of the upper fractions of the hLF and bLF exceeds approximately 95% as determined by the RIA (ie, less than approximately 5% cross-contamination). The resolution of the bLF hLF was somewhat better with the stepped elution than with the linear gradient elution. The purified hLF was added to the raw milk coil and used to determine the chromatographic purification of the bLF hLF in bovine milk using a strong cation exchange resin. Bovine milk to which sodium phosphate has been added, pH 7.5 (20mM), NaCl (0.4M), Tween-20 (0.02%) and either hLF (100 μg / ml) or regulator alone, was stirred for 20 minutes at room temperature (the final pH was 6.6). The skim milk (obtained by centrifugation at 15,000 x g for 30 minutes at 4 ° C) was adjusted to a pH of 4.7 with IN of HCl and incubated at 40 ° C for 30 minutes. The serum fraction (obtained by centrifugation at 15,000 xg for 30 minutes at 4 ° C) was adjusted to a pH of 7.5 with IN of NaOH, and further clarified by centrifugation at 15,000 xg for 5 minutes at 20 ° C followed by filtration. through a 0.22 μm filter. 1 ml samples were applied to the Mono S ™ column equilibrated with 0.4M NaCl, 20mM sodium phosphate, at a pH of 7.5. The column was then rinsed with 18 ml of 0.4 M NaCl, 20 mM sodium phosphate, at a pH of 7.5 to 1 ml / min. The peaks monitored by measuring absorption at 280 nm (total scale of 0.01). Fig. 5 shows the chromatograms when a linear salt gradient is subsequently applied from 0.4-l.OM NaCl in 18 ml of 20 mM sodium phosphate, at a pH of 7.5; panel A shows bovine serum only with bLF (not omitted) and panel B shows bovine serum containing bLF and hLF (omitted). Figure 6 shows the chromatograms when the salt concentration was stepwise increased from 0.4M to 0.6M NaCl in 20mM sodium phosphate, at a pH of 7.5; after 10 minutes, another step increment was applied from 0.6M to 1M NaCl in 20mM sodium phosphate, at a pH of 7.5; panel A shows bovine serum only with bLF (not omitted) and panel B shows bovine serum containing bLF and hLF (omitted). The resolution of the bLF hLF with stepwise elution was better than that observed with a linear salt gradient under the conditions examined. The volume, as well as the salt resistance of an elution buffer required to initiate and establish the complete elution of the hLF are related to the amount of hLF bound to the column. A small increase (eg, staggered from 0.4M to 0.5M NaCl) in the salt concentration will easily and preferentially initiate the elution of the hLF when the column is loaded with increasing amounts of hLF. It was observed that the more hLF is bound to the resin, the lower the concentration of salt required to initiate the elution of the hLF will be and the greater the residue of the hLF peak that occurred. Effects of Salt Concentration on the Elution Volume 100 μg of hLF was loaded onto a Mono column S and variant NaCl concentration elution regulators were applied in 20mM sodium phosphate, at a pH of 7. 5, to completely elute the linked hLF. Table 9 shows the volumes (in ml) of each of the salt concentrations required for complete elution of the hLF. TABLE 9 Dependence of the Elution Volume of the Salt Concentration Volume (ml) required for the complete NaCl (M) elution 0.4 165 0.5 17 0.6 4.9 0.7 2.5 0.8 1.7 0.9 1.5 With the lower salt concentrations, a decrease of the sharpness of eluted peaks. These results indicated that it should be limited the volume of rinsing regulator (eg 0.4M NaCl) during large-scale purification, since rinsing the resin with large volumes relative to the volume of resin packaged under the conditions examined would completely elute the bound hLF. Up-scale purification of hLF Variant amounts of hLF were loaded onto a 1 ml Mono S TM column and a linear salt gradient was applied (0-1% OM NaCl in 30 ml of 20 mM phosphate). sodium, at a pH of 7.5) to ml / min. The concentration of NaCl was recorded in which it was observed that the elution of the hLF began as well as that in which the elution of the hLF was completed. Table 10 shows the results.

TABLE 10 Elution Patterns of Variant Quantities of hLF Linked to a Mono Column S T ~ M Figure 7 illustrates the relationship between the amount of hLF bound to a 1 ml Mono S TM column and the concentration of NaCl at which the elution of hLF was observed when a linear salt gradient was applied (0-1.0 M NaCl in 30 ml of 20 mM sodium phosphate, at a pH of 7.5) to ml / min. The data in Figure 7 gave an indication of the maximum bonding capacity (mg of hLF bound per ml of resin) of Mono S TM. Experiments with Fast Flow of S Sepharose ™ yielded similar results. Affinity chromoatography in Concanavalin A The differences in glycosylation that exist between hLF and bLF can be used to separate the hLF of bLF by lectin chromatography. The hLF bound to Con A could be eluted completely with 50mM of a-methyl-D-mannopyranozide in 50mM of Tris HCl, at a pH of 8.0, 0.14M NaCl, as long as the bLF did not elute significantly, still when 200mM of oc-methyl-D mannopyranozide was used. The chromatography of Con A can thus be used to separate the hLF from the bLF, in such a way that the removal of the bLF residues in the hLF preparations is done by strong cation exchange chromatography, ie, an alternative to the rechromatography in a strong cation exchange resin. Examples of strong cation exchange resins include, but are not limited to: RESIN SUPPLIER S Sepharose Fast Flow Pharmacia SP Sephadex C-50 Pharmacia SP Sephadex C-25 Pharmacia Mono S ™ Pharmacia SP Sepharose Fast Flow Pharmacia SP Sepharose Big Beads Pharmacia A9 50 W X2 BioRoad A9 50 X4 BioRoad A9 50 X8 BioRoad A9 50 W X12 BioRoad A9 50 X16 BioRoad Protein Pa SP 15 HR Millipore / Waters Protein Pak SP 40 HR Millipore / Waters Parcosi1 PepKat Serva Parcomer PekKat Serva Fractogel EMD S03 650 (M) Merck Separation of hLF and bLF with HIC Hydrophobic interaction chromatography (HIC) separates proteins based on differences in Hydrophobic surfaces in molecules. The matrix contains a hydrophobic ligand (similar to a phenyl or butyl group). In the presence of high salt concentrations (eg, >; 1M (NH4) 2S04), the hydrophobic surfaces in the molecules are exposed and bound to the resin. The proteins are eluted by decreasing the salt concentration. It has been reported by Yoshida (J. Diary Sci. 72 (1989) 1446-1450) that bLF can bind to a 650M Toyopearl butyl column. The bLF is not eluted using deionized water, but by using 0.25M acetic acid. It was also reported that the LF of human tears is linked to the HIC columns (Baier et al. (1990) J. Chromat, 525, 319-328). By using the HPLC of the reverse phase (phenyl), Hutchens et al. (PNAS USA 88 (1991) 2994-2998) showed that the human LF and the degradation products of the LF can be separated. The tryptic fragments of human LF could be separated with reverse phase HPLC (octadecyl) (Shimazaki et al. (1993) J. Diary Sci. 76, 946-955). The prior art does not suggest that hLF and bLF can be separated with HCl. In accordance with the above, the invention includes the use of HCl in the separation of hLF and bLF and the corresponding methods. 500 μg of pure hLF or bLF, diluted in regulator A (50 mM NaPi at a pH of 7.5, 2.5 M (NH4) 2S04), in a 1 ml column of PF Sepharose FF (Pharmacia, - sub elevated) at a flow rate of 0.2 ml / min. After a 5 ml rinse with regulator A, all of the hLF was eluted in block form with approximately 11 ml of 80% of the regulator (regulator B = 50 mM NaPi at a pH of 7.5). No elution of bLF was observed under these conditions. All bLF was eluted with a subsequent block (with 5 ml) of buffer B at 100%. This result clearly showed that hLF and bLF can be separated with this technique. One disadvantage is the relatively high salt concentrations in which the proteins are eluted. The technique can be used to further separate the transgenic hLF from the bLF obtained after S Sepharose chromatography. Mouse Domferrin Another aspect of the invention is a separate mouse domferrin protein having the N-terminal sequence lys-ala-val-arg-val-gln-trp-xxx-ala-val-ser-asn-glu-glu , a very approximate molecular weight by SDS PAGE of 80 kD, and an elution profile of Mono S by which the protein is obtained in a concentration of approximately 0.22M salt. The invention includes the preparation of such proteins by cation exchange chromatography.

Some of the preferred properties of mouse domferrin can be summarized: 1. It is eluted from Mono S in a single position (ie, 0.22M salt, mouse lactoferrin is eluted at 0.26M salt). 2. The protein has a unique N-terminal sequence, different from mouse lactoferrin, and different from mouse transferrin (and other lactoferrin and transferrin species) - see tables above. 3. The migration pattern is indicated on the SDS-PAGE and -80 kD of protein. (Confused bands are observed, probably due to differences in glycosylation). Additional Information: 4. Immunodiffusion assays show no cross-reactivity of mouse domferrin with anti-mouse transferrin or anti-human lactoferrin antibodies. 5. Mouse domferrin has no peroxide activity (tested in a lactoperoxidase assay). Although the present invention has been described in some details by way of illustration for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the claims.

Claims (34)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and therefore the property described in the following claims is claimed as property. A method for substantially purifying human lactoferrin from milk, comprising the steps of: contacting the milk or milk fraction containing human lactoferrin with a strong cation exchange resin under high ionic strength conditions for a period of time adequate bonding period which forms human lactoferrin-resin complexes; removing milk or milk fraction that remains unbound and recovering the human lactoferrin-resin complexes, and eluting the human lactoferrin complexes recovered from human lactoferrin resin with a salt solution having sufficient ionic strength to elute human lactoferrin from human lactoferrin-resin complexes. A method according to claim 1, characterized in that said step of contacting said milk or fraction of the milk with said strong cation exchange resin is conducted with a limiting amount of said strong cation exchange resin having a capacity of of binding approximately equal to or less than the expected amount of human lactoferrin in milk or
2. fraction of milk.
3. 3. A method according to claim 1, characterized in that the milk or milk fraction is adjusted to approximately 0.35-0.4M salt by the addition of NaCl or KCl before contacting the milk or fraction of the milk with said resin of strong cation exchange.
4. 4. A method according to claim 1, characterized in that the transgenic bovine milk serum is the fraction of milk used to contact said resin with strong cation exchange.
5. A method according to claim 1, characterized in that it comprises the additional intermediate step of rinsing the recovered human lactoferrin-resin complexes with a rinse buffer before eluting the human lactoferrin.
6. 6. A method according to claim 5, characterized in that the complexes recovered from human lactoferrin resin are spilled onto a column.
7. 7. A method according to claim 1, characterized in that the resin of strong exchange of

   • ** * TM cation is selected from the group consisting of Mono S and TM

   S Sepharose.
8. 8. A method according to claim 1,

   characterized in that the salt solution for eluting the human lactoferrin is at least 0.4M NaCl or 0.4M KCl.
9. 9. A method according to claim 1, characterized in that human lactoferrin is eluted by step elution comprising an elution step having a salt solution that substantially elutes human lactoferrin without substantially eluting bovine lactoferrin.
10. A method according to claim 7, characterized in that the human lactoferrin is eluted and separated from the bovine lactoferrin by a salt gradient.
11. A method according to claim 1, characterized in that it comprises the additional step of recovering the human lactoferrin eluted, loading the human lactoferrin recovered in a resin of strong exchange of cation or resin A of concanavalin, and eluting the human lactoferrin of the resin in substantially purified form.
12. 12. A method according to claim 1, characterized in that the step of contacting the milk or milk fraction is carried out through a dialysis membrane that is substantially permeable to human lactoferrin.
13. 13. A method according to claim 1, characterized in that the high ionic strength conditions are produced by the addition of exogenous NaCl or KCl and / or a regulator and / or a detergent to the milk or fraction of the milk to which it has or is not a non-ionic detergent has been added.
14. 14. A method for purifying native human lactoferrin from the degradation products of hLF in a sample, comprising the step of contacting the sample with a strong cation exchange resin under suitable binding conditions and eluting the native human lactoferrin as a soluble peak by applying a gradient of salt or stepped elution and recovering the peak consisting of essentially of native human lactoferrin having an intact amino terminal sequence.
15. 15. A method according to claim 14, characterized in that the degradation products comprise human lactoferrin species lacking amine terminal amino acids in comparison to the encoded human lactoferrin protein.
16. 16. A method according to claim 14, characterized in that the degradation products are eluted at a lower salt concentration at which the native human lactoferrin is eluted.
17. 17. A method to separate lactoferrin

    of the bovine lactoferrin in a sample, comprising contacting the sample with a strong cation exchange resin and eluting the human lactoferrin from the resin with a salt solution that preferentially elutes human lactoferrin but does not substantially elute lactoferrin bovine concomitantly with human lactoferrin.
18. 18. A method according to claim 17, characterized in that the hLF and the bLF are separated by staggered increments of the salt concentration.
19. 19. A method according to claim 17, characterized in that the hLF and the bLF are separated by elution with a salt gradient.
20. 20. A method according to claim 17, characterized in that the resin of strong exchange of • JT? j, TM cation is Mono S or Rapid Sepharose S, suitable binding conditions comprise high ionic strength, and elution of human lactoferrin is carried out with a salt solution that elutes preferentially human lactoferrin but does not elute substantially non-human lactoferrin concomitantly with human lactoferrin.
21. 21. A method for separating substantially pure recombinant human lactoferrin from the milk of a transgenic animal harboring a transgene encoding the

    expression and secretion of recombinant human lactoferrin in its milk, comprising the steps of: obtaining milk or a milk fraction of said transgenic animal containing recombinant human lactoferrin; contacting said milk or milk fraction with a strong cation exchange resin under suitable binding conditions for the binding of human lactoferrin to the resin; elute the recombinant human lactoferrin from the strong cation exchange resin with a graduated salt gradient or a linear salt gradient.
22. 22. A method according to claim 21, characterized in that the resin of strong exchange of cation • or - ** n is Mono STM and S Sepharose ™, the conditions of which are: Suitable linkers comprise the high ionic strength, and the elution of human lactoferrin is carried out by eluting with a salt solution of at least 0.5M NaCl.
23. 23. A method according to claim 21, characterized in that suitable binding conditions are the milk or milk fraction adjusted to approximately 0.35-0.4M NaCl and the performance of the elution with a salt solution consisting essentially of at least 0.4. M NaCl or KCl.
24. 24. Recombinant human lactoferrin produced in the milk of a transgenic non-human animal and substantially purified from said milk by the

    strong cation exchange chromatography and / or concanavalin A resin.
25. 25. A method for removing the LPS from a solution comprising the step of contacting a solution containing LPS with a resin comprising immobilized hLF and recovering the portion of the solution that binds to the resin comprising the immobilized hLF.
26. 26. A method according to claim 24, characterized in that the resin comprising the immobilized hLF is hLF linked covalently to the Sepharose.
27. A method according to claim 1, characterized in that the human lactoferrin is purified from milk or a milk fraction containing bovine lactoferrin, by which the human lactoferrin is separated from the bovine lactoferrin by differential elution with a solution of Salt .
28. 28. The use of hydrophobic interaction chromatography in the separation of human lactoferrin from bovine lactoferrin.
29. 29. A method for the separation of human and bovine lactoferrin, which comprises attaching a mixture including said lactoferrins to a hydrophobic interaction chromatography, and eluting human lactoferrin separately from bovine lactoferrin.
30. 30. A mouse-isolated domferrin protein having the N-terminal sequence lys-ala-val-arg-val-gln-trp-xxx-ala-val-ser-asn-glu-glu, a molecular weight very close to the SDS 80 kD PAGE, and an elution profile of Mono S by which the protein is obtained in a concentration of approximately 0.22M salt.
31. 31. A mouse isolated domferrin protein according to claim 30, characterized in that it does not exhibit cross-reactivity with the anti-mouse transferrin or with anti-human lactoferrin antibodies.
32. 32. A mouse isolated domferrin protein according to claim 30 or 31, characterized in that it has no peroxidase activity.
33. 33. A method for the purification of a mouse domferrin protein comprising attaching a source of said domferrin to cation exchange chromatography.
34. 34. The use of cation exchange chromatography when purifying mouse domferrin.