Lactoferrin Compositions and Methods of Use

Abstract

Compositions comprising lactoferrin, particularly pharmaceutical formulations comprising lactoferrin purified from raw natural milk sources, are provided, as are methods of making and using the same.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 304,434, filed January 28, 2022, and U.S. Provisional Application No. 63 / 236,204, filed August 23, 2021, the entire contents of each of which are incorporated by reference herein for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created in XX month, 20XX, is named XXXXXUS_sequencelisting.txt and is X,XXX,XXX bytes in size. [Background technology]

[0003] Lactoferrin has been explored in terms of its antibacterial properties. However, preparations of lactoferrin are usually derived from pre-processed and / or pre-treated dairy products, such as pasteurized milk sources, or are recombinantly produced. Such production strategies can result in altered properties of lactoferrin compared to those found in raw and / or untreated milk sources, including denaturation, reduced biological activity, reduced iron-binding capacity, altered glycosylation, and / or lack of retention of post-translational modifications. Furthermore, available purified lactoferrin products generally contain significant impurities.

[0004] There are no preparations and formulations of lactoferrin in the art that are directed to retaining the natural biological activity of lactoferrin, including lactoferrin that is generally free of impurities (e.g., other proteins, enzymes, endotoxins, prions, etc.) and that are pharma- ceutically acceptable (e.g., FDA registrable) and particularly suitable for use in the areas of wound care and other applications. Summary of the Invention

[0005] Provided herein is a pharmaceutical composition comprising lactoferrin. Also provided herein is a composition comprising lactoferrin.

[0006] In some embodiments, a pharmaceutical composition comprising lactoferrin is provided herein, and the pharmaceutical composition is formulated for delivery to the oral cavity or nasal cavity.In some embodiments, the pharmaceutical composition is formulated for retention of lactoferrin in the buccal mucosa.In some embodiments, the pharmaceutical composition is formulated for delivery to the extracellular matrix (ECM) of cells.

[0007] In some embodiments, the pharmaceutical composition is formulated as a tablet. In some embodiments, the tablet is formulated as an orally dissolving or disintegrating tablet.

[0008] In some embodiments, the pharmaceutical composition is formulated as a nasal spray.

[0009] In some embodiments, the pharmaceutical composition is formulated as a gum.

[0010] In some embodiments, the pharmaceutical composition is formulated as an oral thin film.

[0011] In some embodiments, the pharmaceutical composition is formulated as a lozenge.

[0012] In some embodiments, the oral or nasal cavity includes the nasopharynx.

[0013] Also provided herein is a pharmaceutical composition comprising lactoferrin, the pharmaceutical composition being formulated for delivery to an open wound or ulcer.In some embodiments, the pharmaceutical composition is formulated for delivery to skin tissue, mucosal tissue, ocular tissue, connective tissue, gastrointestinal tissue and / or pulmonary tissue.In some embodiments, the wound comprises an open wound or ulcer.In some embodiments, the ulcer is venous or arterial.

[0014] In some embodiments, the wound is selected from the group consisting of a burn, a bite, a cut, an infection, an ulcer, and combinations thereof. In some embodiments, the burn is selected from the group consisting of a thermal burn, a chemical burn, an electrical burn, a radiation burn, a friction burn, and combinations thereof. In some embodiments, the cut is selected from the group consisting of a puncture wound, a laceration, an abrasion, an incision (which may be surgical in nature), a cut, and combinations thereof. In some embodiments, the ulcer is venous or arterial.

[0015] In some embodiments, the pharmaceutical composition comprises a bioadhesive.

[0016] In some aspects, the pharmaceutical composition is formulated in a delivery format selected from the group consisting of a gel foam, a hydrogel, an injection dressing, a powder, a spray, and an inhalation format.

[0017] Also provided herein is a pharmaceutical composition comprising lactoferrin, which is formulated for delivery to the digestive tract.In some aspects, the pharmaceutical composition is formulated in a delivery format comprising microencapsulation.In some aspects, the pharmaceutical composition is formulated in a delivery format comprising enteric coating.

[0018] Also provided herein is a composition comprising lactoferrin, the composition being formulated for delivery to the oral cavity, nasal cavity, wound, ocular cavity, and / or gastrointestinal tract. In some embodiments, the wound comprises an open wound or ulcer. In some embodiments, the ulcer is venous or arterial.

[0019] In some embodiments, the pharmaceutical composition or compositions comprises an excipient.

[0020] In some embodiments, the pharmaceutical composition or composition comprises sodium steryl humate, carbomer, sodium hydroxide, glycerin, sodium benzoate, benzoic acid, citric acid, and / or combinations thereof. In some embodiments, the pharmaceutical composition or composition comprises xylitol, sorbitol, mannitol, maltitol, sugar alcohol, sucralose, and / or combinations thereof. In some embodiments, the pharmaceutical composition or composition comprises isomalt, microcrystalline cellulose, sodium carboxymethylcellulose, and / or combinations thereof. In some embodiments, the pharmaceutical composition or composition comprises powdered sugar, gum base, corn syrup, gum arabic, and / or combinations thereof. In some embodiments, the pharmaceutical composition or composition comprises HiG PWD-04, encapsulated powdered flavor, anhydrous or natural caffeine, encapsulated sucralose or sucralose, encapsulated acesulfame-K and aspartame, aspartame, acesulfame-K, triacetin, silicon dioxide, and / or combinations thereof.

[0021] In some embodiments, the pharmaceutical composition or compositions include a clotting agent, hi some embodiments, the clotting agent includes thrombin, amylopectin, kaolin, and / or combinations thereof.

[0022] In some embodiments, the pharmaceutical composition or compositions include an astringent, hi some embodiments, the astringent is selected from the group consisting of alum, acacia, sage, yarrow, witch hazel, bayberry, distilled vinegar, persimmon, green tea, black tea, citric acid, cranberry juice and / or extract, grapefruit juice and / or extract, inorganic acids, and combinations thereof.

[0023] In some embodiments, the lactoferrin is bovine. In some embodiments, the lactoferrin is untreated. In some embodiments, the lactoferrin is not chemically, enzymatically, acidically, and / or heat treated. In some embodiments, the lactoferrin is not heat treated. In some embodiments, the lactoferrin is not heat treated at a temperature of 50° C. or higher, 51° C. or higher, 52° C. or higher, 53° C. or higher, 54° C. or higher, or 55° C. or higher. In some embodiments, the lactoferrin is not heat treated at a temperature of 55° C. or higher.

[0024] In some aspects, the purified lactoferrin comprises a native conformation as assessed by circular dichroism.

[0025] In some embodiments, the purified lactoferrin comprises a native conformation as assessed by differential scanning calorimetry (DSC). In some embodiments, the native conformation comprises an apolactoferrin conformation and / or a hololactoferrin conformation. In some embodiments, the apolactoferrin conformation has a peak melting temperature of 60.2±0.8° C. and / or the hololactoferrin conformation has a peak melting temperature of 88.38±0.8° C.

[0026] In some embodiments, purified lactoferrin can bind iron. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of purified lactoferrin can bind iron. In some embodiments, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of purified lactoferrin can bind iron. In some embodiments, at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% of purified lactoferrin can bind iron. In some embodiments, the ability to bind iron is evaluated by DSC.

[0027] In some embodiments, the purified lactoferrin comprises a post-translational modification. In some embodiments, the post-translational modification comprises glycosylation.

[0028] In some embodiments, the purified lactoferrin comprises an average molecular weight of at least 79,000 to 86,000 Da.

[0029] In some embodiments, the purified lactoferrin is dried. In some embodiments, the purified lactoferrin is dried by freeze-drying / lyophilization, fluidized bed drying, or low temperature spray drying. In some embodiments, the purified lactoferrin remains in liquid form throughout lactoferrin purification.

[0030] In some embodiments, the composition further comprises an iron molecule. In some embodiments, the purified lactoferrin is complexed with an iron molecule. In some embodiments, the iron molecule comprises Fe2+ or Fe3+. In some embodiments, the purified lactoferrin is complexed with a copper, zinc, manganese, and / or gallium molecule. In some embodiments, the purified lactoferrin is complexed with a zinc molecule.

[0031] In some embodiments, the composition contains endotoxin at a level of 5 EU / kg or less.

[0032] In some embodiments, lactoferrin is purified from unprocessed natural dairy products. In some embodiments, the natural dairy products are not processed prior to purification of lactoferrin. In some embodiments, the natural dairy products are not chemically, enzymatically, acidically, or thermally treated prior to purification of lactoferrin. In some embodiments, the natural dairy products are not heat treated prior to purification of lactoferrin. In some embodiments, the heat treatment comprises a temperature of 50°C or higher, 51°C or higher, 52°C or higher, 53°C or higher, 54°C or higher, or 55°C or higher. In some embodiments, the heat treatment comprises a temperature of 55°C or higher.

[0033] In some embodiments, lactoferrin is purified from a natural dairy product that has been separated into skim milk and cream prior to purification of lactoferrin. In some embodiments, the separation into skim milk and cream comprises cold bowl separation. In some embodiments, lactoferrin is purified from a natural dairy product that has been acid treated prior to purification of lactoferrin. In some embodiments, the acid treatment comprises removal of insoluble casein. In some embodiments, the acid treatment is performed at a pH of 4.0 or greater.

[0034] Also provided herein is a method for producing any one of the pharmaceutical compositions or compositions provided herein. In some embodiments, the method comprises one or more steps selected from the group consisting of chromatography, filtration, and drying. In some embodiments, the method comprises each of the steps of chromatography, filtration, and drying.

[0035] Also provided herein are methods of treating a disease or condition comprising administering any one of the pharmaceutical compositions or compositions provided herein. In some embodiments, the disease or condition is selected from the group consisting of a pathogenic disease, a gastrointestinal (GI) infection, a wound infection, an allergic condition, an inflammatory condition, and combinations thereof.

[0036] Also provided herein is a method of treating a pathogenic disease, the method comprising administering to a subject any one of the pharmaceutical compositions or compositions described above.

[0037] In some embodiments, the administration is prophylactic. In some embodiments, the subject is at risk of exposure to a pathogen. In some embodiments, the subject has been exposed to a pathogen. In some embodiments, the subject has been diagnosed with an infection by a pathogen.

[0038] Also provided herein is a method for reducing the risk of pathogenic disease, comprising administering to a subject any one of the pharmaceutical compositions or compositions provided herein. In some embodiments, the risk of pathogenic disease comprises administering to a wound of the subject a pharmaceutical composition or composition. In some embodiments, the subject has been exposed to a pathogen and / or has been diagnosed with an infection caused by a pathogen.

[0039] In some embodiments, the pathogen comprises an orally and / or nasally transmitted microorganism. In some embodiments, the pathogen comprises an airborne microorganism.

[0040] In some embodiments, the pathogen comprises a virus. In some embodiments, the virus comprises a coronavirus. In some embodiments, the coronavirus is SARS-CoV-2.

[0041] In some embodiments, the pathogen comprises a bacterium.

[0042] In some embodiments, the pathogen comprises a fungus.

[0043] In some embodiments, treating a disease or condition includes prophylactic treatment of the disease or condition.

[0044] Also provided herein is a method for reducing the risk of infection, comprising administering to a subject any one of the above pharmaceutical compositions or compositions. In some embodiments, the subject has a wound that is at risk of infection.

[0045] Also provided herein is a method of promoting wound healing of tissue, comprising administering any one of the pharmaceutical compositions or compositions described above to a wound in a tissue of a subject. In some embodiments, the wound is selected from the group consisting of a burn, a cut, an infection, an ulcer, and combinations thereof. In some embodiments, the burn is selected from the group consisting of a thermal burn, a chemical burn, an electrical burn, a radiation burn, a friction burn, and combinations thereof. In some embodiments, the cut is selected from the group consisting of a puncture wound, a laceration, an abrasion, an incision, a cut, and combinations thereof. In some embodiments, the tissue is a skin tissue, a lung tissue, or combinations thereof. In some embodiments, the method comprises treating a disease or condition selected from the group consisting of a pathogenic disease, a gastrointestinal (GI) infection, a wound infection, an allergic condition, an inflammatory condition, and combinations thereof. In some embodiments, treating a disease or condition comprises prophylactic treatment of a disease or condition. [Brief description of the drawings]

[0046] [Figure 1] A specific embodiment of the lactoferrin purification method described herein is shown. Briefly, raw milk (e.g., untreated milk that has not been chemically, enzymatically, acidically, or thermally treated before lactoferrin purification) is (1) diverted before entering the industry standard milk processing workflow, (2) run through an ion exchange resin / column, the effluent is usually (e.g., if desired / required by the raw milk producer) returned to the standard milk processing workflow, the lactoferrin-containing eluate is collected, (3) the collected eluate is filtered, and (4) the purified lactoferrin is processed.

[0047] [Figure 2A] Figure 1 shows MALDI-TOF (approximately 18 kDa-100 kDa) purity assessment of lactoferrin produced according to Example 1. The raw material used was raw colostrum (RC).

[0048] [Figure 2B]Figure 1 shows MALDI-TOF (approximately 18 kDa-100 kDa) purity assessment of lactoferrin produced according to Example 1. The raw material used was raw whole milk (RM).

[0049] [Figure 3A] MALDI-TOF (approximately 18 kDa to 100 kDa) purity assessment of over-the-counter (OTC) lactoferrin supplements is shown.

[0050] [Figure 3B] MALDI-TOF (approximately 18 kDa to 100 kDa) purity assessment of purchased laboratory reagent grade lactoferrin is shown.

[0051] [Figure 3C] MALDI-TOF (approximately 18 kDa to 100 kDa) purity assessment of purchased laboratory reagent grade lactoferrin is shown.

[0052] [Figure 4] 1 shows Orbitrap Velos mass spectrometry purity assessment of lactoferrin produced according to Example 1.

[0053] [Figure 5A] FIG. 1 shows an ELISA purity assessment of lactoferrin produced according to Example 1 and purchased laboratory reagent grade lactoferrin. [Figure 5B] FIG. 1 shows an ELISA purity assessment of lactoferrin produced according to Example 1 and purchased laboratory reagent grade lactoferrin.

[0054] [Figure 6] FIG. 1 shows lactoferrin enrichment in the ECM by immunofluorescence (IF) imaging.

[0055] [Figure 7] Quantification of lactoferrin enrichment in ECM.

[0056] [Figure 8]FIG. 1 shows lactoferrin enrichment in the ECM by immunofluorescence (IF) imaging in primary oral cells.

[0057] [Figure 9] 1 shows a CPE reduction assay of SARS-CoV-2 in the presence of API-E2.

[0058] [Figure 10] FIG. 1 shows a differential scanning calorimetry (DSC) assay plot evaluating API-E2 lactoferrin (10 mg / ml), including the heat treated version.

[0059] [Figure 11A] 1 shows a DSC assay plot evaluating API-E2 lactoferrin in the absence and presence of excess iron.

[0060] [Figure 11B] Shown is a DSC assay plot evaluating laboratory grade standard lactoferrin and commercial supplement grade lactoferrin overlaid on the plot for API-E2 lactoferrin (see FIG. 10).

[0061] [Figure 12] FIG. 1 shows the evaluation of the biological activity of lactoferrin against salivary bacteria as an assessment of pH over time in the presence of API-E2.

[0062] [Figure 13] Photographs are shown of untreated poultry (left) or API-E2-treated poultry (right) that were inoculated with human salivary bacteria and incubated at 37°C for six days.

[0063] [Figure 14] 1 shows a zone of inhibition study performed to evaluate the biological activity of API-E2 lactoferrin against E. coli.

[0064] [Figure 15]1 shows a summary of lactoperoxidase (LPO) activity and results for various lactoferrin samples containing API-E2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] definition Terms used in the claims and specification are defined as set forth below, unless otherwise stated.

[0066] As used interchangeably herein, "raw dairy product" or "raw lactoferrin-containing dairy product" refers to the natural liquid milk product produced by the mammary gland of a mammal and taken directly from the mammal without any additional processing (e.g., filtration, column / resin purification, and / or milk separation) and / or treatment steps (e.g., chemical treatment, enzymatic treatment, acid treatment, and / or heat treatment) being applied.

[0067] As used interchangeably herein, "unprocessed dairy product" or "unprocessed lactoferrin-containing dairy product" refers to a lactoferrin-containing dairy product that has not undergone processing steps (e.g., chemical, enzymatic, acid, and / or heat treatment) but has potentially undergone one or more mechanical processing steps (e.g., filtration, column / resin purification, and / or milk separation).

[0068] Unless specifically stated or otherwise clear from the context, the term "about" as used herein is understood to be within the normal tolerance in the art, for example, within 2 standard deviations of the mean. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Numerical values ​​provided herein may be considered to be modified by the term about. In this case, it is clear from the context that the range encompassed by this modification is consistent with the operability of the invention and the clarity of the claims.

[0069] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA, from the Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Dr., Madison, Wis.)), or by visual inspection (see generally, Ausubel et al.).

[0070] One example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0071] Lactoferrin Compositions comprising purified lactoferrin are provided herein. Generally, lactoferrin herein refers to a purified form of mammalian lactoferrin obtained from unprocessed dairy products (e.g., raw milk).

[0072] The lactoferrin of the purified composition herein is typically derived from bovine sources, such as from milk sources.Exemplary bovine lactoferrin (bLF) molecules include, but are not limited to, those described in CAS Registry No. 146897-68-9, and Mead and Tweedie (Nucleic Acids Res. 1990 Dec 11;18(23):7167.) and Pierce et al. (Eur J Biochem. 1991 Feb 26;196(1):177-84.), each of which is incorporated herein by reference for all purposes.The non-limiting full-length amino acid sequence of exemplary bovine (Bos taurus) lactoferrin is provided below.(SEQ ID NO: 1; GenBank Accession No. AAA30610.1).

[0073] Lactoferrin may refer to a fragment of full-length lactoferrin (e.g., SEQ ID NO: 1). Fragments include biologically active fragments. As used herein, "biologically active" refers to a protein that has one or more of the biological activities of the corresponding native protein. Examples of biological activities include, but are not limited to, enzymatic activity, antimicrobial activity (e.g., antibacterial, antifungal, and / or antiviral activity), iron binding / sequestering activity, immunomodulatory effects (e.g., anti-inflammatory activity), growth regulation, and cell surface affinity, wound healing, or any of the other activities of lactoferrin described herein or known in the art. For example, lactoferrin is typically secreted, and biologically active fragments may include secreted forms, such as "processed" fragments of lactoferrin that lack the signal peptide. As an illustrative example, lactoferrin represented by SEQ ID NO: 1 includes the signal peptide MKLFVPALLSLGALGLCLA (SEQ ID NO: 2; amino acids 1-19 of SEQ ID NO: 1). Thus, a biologically active fragment of lactoferrin can include lactoferrin lacking the signal peptide (eg, amino acids 20-708 of SEQ ID NO:1).

[0074] Lactoferrin may refer to lactoferrin isoforms such as lactoferrin alpha (LFα), lactoferrin beta (LFβ), or lactoferrin gamma (LFγ) isoforms.

[0075] Lactoferrin may have an amino acid sequence at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 1 or a biologically active fragment thereof (e.g., a secreted form such as amino acids 20-708 of SEQ ID NO: 1 lacking the signal peptide). Lactoferrin may have an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1 or a biologically active fragment thereof. Lactoferrin may have an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 1 or a biologically active fragment thereof. Lactoferrin may have an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 1 or a biologically active fragment thereof. Lactoferrin may have an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 1 or a biologically active fragment thereof. Lactoferrin may have an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1, or a biologically active fragment thereof. Lactoferrin may have an amino acid sequence that is at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO:1, or a biologically active fragment thereof.

[0076] Lactoferrin may have conservative substitution. "Conservative substitution" or "conservative amino acid substitution" refers to the replacement of amino acid with chemically or functionally similar amino acid.Conservative substitution is well known in the art (e.g., as described in Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) WH Freeman&Co., New York, NY), and is incorporated herein by reference for all purposes.

[0077] Post-translational modifications Lactoferrin contained in raw dairy products as raw materials usually contains post-translational modifications.Without wishing to be bound by theory, the purification strategies and methods described herein are designed to reduce, minimize, or eliminate the destruction of native post-translational modifications (with reference to those contained in the native milk source used for the purification of lactoferrin).For example, the purification strategies and methods described herein reduce, minimize, or eliminate chemical treatment, enzyme treatment, acid treatment, heat treatment (e.g., pasteurization), and / or any other treatment that can destroy native post-translational modifications.

[0078] Post-translational modifications may refer to those modifications that are involved in the biological activity of lactoferrin. Generally, recombinantly produced lactoferrin is typically produced in an exogenous expression system such as bacteria or yeast, and lacks the post-translational and / or post-translational modifications of naturally produced lactoferrin. Post-translational modifications include, but are not limited to, glycosylation, phosphorylation, and acetylation. In particular, post-translational modifications may include glycosylation (e.g., N-linked glycosylation), such as glycosylation at asparagine 233, 281, 368, 476, and / or 545, which may include -acetylneuraminic acid, galactose, mannose, fucose, N-acetylglucosamine, and / or N-acetylgalactosamine. Post-translational modifications may refer to naturally occurring and / or non-naturally occurring (e.g., modifications after purification, such as by in vitro methods known to those skilled in the art). Post-translational modifications include processing of full-length lactoferrin, such as removal of signal sequences as described above.

[0079] Unmodified secreted lactoferrin typically has a molecular weight of about 78 kDa. Natural post-translational modifications may result in molecular weights in the range up to about 86 kDa. Purified lactoferrin may have a molecular weight greater than about 78 kDa. Purified lactoferrin may have a molecular weight of at least 79 kDa. Purified lactoferrin may have a molecular weight of at least 80 kDa. Purified lactoferrin may have a molecular weight of at least 81 kDa. Purified lactoferrin may have a molecular weight of at least 82 kDa. Purified lactoferrin may have a molecular weight of at least 83 kDa. Purified lactoferrin may have a molecular weight of at least 84 kDa. Purified lactoferrin may have a molecular weight of at least 85 kDa. Purified lactoferrin may have a molecular weight of at least 86 kDa. Purified lactoferrin may have a molecular weight of 79-86 kDa. Purified lactoferrin may have a molecular weight of 82-84 kDa. The purified lactoferrin may have a molecular weight of 82-85 kDa.

[0080] Methods for assessing post-translational modifications are known to those of skill in the art and include mass spectrometry or antibody-mediated methods (e.g., the use of antibodies that recognize post-translational modifications such as ELISA and / or two-dimensional Western blot analysis).

[0081] Protein structure The conformational state of lactoferrin contained in raw dairy products is generally considered to be the natural conformation of lactoferrin. It is generally believed that various treatments of milk sources that are usually used to purify lactoferrin (such as pasteurization) can denature proteins. Without wishing to be bound by theory, the purification strategies and methods described herein are designed to reduce, minimize, or eliminate the destruction of the natural conformational state (referring to the conformational state found in the natural milk sources used to purify lactoferrin). For example, the purification strategies and methods described herein reduce, minimize, or eliminate chemical treatment, enzyme treatment, acid treatment, heat treatment (e.g., pasteurization), and / or any other treatment that can denature lactoferrin.

[0082] Methods for assessing conformational states are known to those of skill in the art and include, for example, circular dichroism, X-ray crystallography, or antibody-mediated methods (e.g., the use of antibodies that recognize conformational states and / or non-denaturing Western blot analysis).

[0083] Iron complexation Lactoferrin contains two iron-binding domains (also called globular lobes). Without wishing to be bound by theory, it is believed that iron-binding properties can mediate and / or influence antimicrobial bioactivity, such as microbial killing, prevention of microbial invasion, chelating, elimination of microorganisms, and / or inhibition of growth.

[0084] Iron-bound lactoferrin is called hololactoferrin and iron-free lactoferrin is called apolactoferrin, and in some cases, the biological activity may differ. Examples include antimicrobial activity or other properties, examples of which include increased resistance of hololactoferrin to heat-induced changes compared to apolactoferrin. Lactoferrin in raw dairy products is usually found within a defined ratio of iron-free to iron-bound forms. For example, lactoferrin in cow's milk is generally present at 20-30% in iron-bound form, and in human milk is generally present at 6-8% in iron-bound form. The purified lactoferrin in the compositions herein may have a defined range of iron-bound hololactoferrin. Purified lactoferrin may refer to 20-30% hololactoferrin. Purified lactoferrin may refer to 6-8% hololactoferrin. Purified lactoferrin may refer to more than 30% hololactoferrin. Purified lactoferrin may refer to less than 6% hololactoferrin. Purified lactoferrin may refer to at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% hololactoferrin. Purified lactoferrin may refer to at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% hololactoferrin. Purified lactoferrin may refer to at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% hololactoferrin. Purified lactoferrin may refer to 100% hololactoferrin. Purified lactoferrin may refer to less than 20% hololactoferrin. Purified lactoferrin may refer to more than 8% hololactoferrin. Purified lactoferrin may refer to less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% hololactoferrin. Purified lactoferrin may refer to less than 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% hololactoferrin. Purified lactoferrin may refer to 100% of the iron-free apolactoferrin form.

[0085] Differential scanning calorimetry (DSC) can be used to assess the ability of purified lactoferrin to form hololactoferrin, for example, the ability of purified lactoferrin to bind iron. For example, the ability to bind iron can be assessed by adding excess iron in the presence of purified lactoferrin, then running DSC to assess the relative peaks associated with apolactoferrin and hololactoferrin. The purified lactoferrin and / or dosage formula can include purified lactoferrin, and at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the purified lactoferrin can bind iron. The purified lactoferrin and / or dosage formula can include purified lactoferrin, and at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the purified lactoferrin can bind iron, as assessed by DSC. The purified lactoferrin and / or dosage formulas can include purified lactoferrin, where at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the purified lactoferrin can bind iron. The purified lactoferrin and / or dosage formulas can include purified lactoferrin, where at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the purified lactoferrin can bind iron as assessed by DSC. The purified lactoferrin and / or dosage formulas can include purified lactoferrin, where at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% of the purified lactoferrin can bind iron. The purified lactoferrin and / or dosage formula can include purified lactoferrin where at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% of the purified lactoferrin is capable of binding iron as assessed by DSC. The purified lactoferrin and / or dosage formula can include purified lactoferrin where 100% of the purified lactoferrin is capable of binding iron.The purified lactoferrin and / or dosage formula can include purified lactoferrin, where 100% of the purified lactoferrin is capable of binding iron as assessed by DSC (e.g., in the presence of excess iron the only peak observable is that associated with hololactoferrin).

[0086] Differential scanning calorimetry (DSC) can be used to assess the melting temperature peak of purified lactoferrin, particularly the melting temperature peak of apolactoferrin and / or hololactoferrin. The purified lactoferrin and / or dosage formula can include purified lactoferrin, and the melting temperature peak associated with apolactoferrin is 60.2±0.8°C. The purified lactoferrin and / or dosage formula can include purified lactoferrin, and the melting temperature peak associated with hololactoferrin is 88.38±0.8°C.

[0087] Lactoferrin in the form of hololactoferrin is usually found in natural milk sources with two ferric iron (Fe 3+ The purified lactoferrin in the compositions herein can bind metal ions other than ferric ions, examples of which include, but are not limited to, copper, zinc, manganese, and / or gallium. The purified lactoferrin can bind zinc ions. The purified lactoferrin can bind Fe 2+ Purified lactoferrin bound to metal ions other than ferric ions can be in any of the hololactoferrin or apolactoferrin forms of ferric bound lactoferrin described herein, for example, in any defined ratio of hololactoferrin forms to apolactoferrin forms described herein.

[0088] The conformational states of lactoferrin, e.g., hololactoferrin and apolactoferrin conformations, are characterized by varying levels of metal ion (e.g., Fe 3+). For example, apolactoferrin is typically saturated with less than 5% iron ions, whereas hololactoferrin is typically about 100% saturated. Bovine-derived lactoferrin in raw dairy products is usually 15-20% iron saturated.

[0089] Methods for controlling the defined ratio of hololactoferrin form to apolactoferrin form are known to those skilled in the art. For example, methods for controlling (such as adding or removing) the concentration of ferric ions, metal ions other than ferric ions, and / or non-ferric-based and / or non-ferric-derived molecules are known in the art (e.g., as described in Majka et al. [Analytical and Bioanalytical Chemistry volume 405, pages 5191-5200 (2013)], which is incorporated herein by reference for all purposes).

[0090] Methods for assessing the ability of purified lactoferrin to bind metal ions, such as chemical assays and / or absorbance spectroscopy, are known to those of skill in the art. For example, without wishing to be bound by theory, processing methods (e.g., methods typically used in industrial purification) and / or recombinant production methods may alter the metal binding ability of lactoferrin (e.g., through denaturation of the iron-binding domain).

[0091] Methods for assessing the ratio of hololactoferrin forms to apolactoferrin forms are known to those of skill in the art, such as chemical assays and / or absorbance spectroscopy (e.g., as described in Majka et al. [Analytical and Bioanalytical Chemistry volume 405, pages 5191-5200 (2013)], incorporated herein by reference for all purposes).

[0092] In a non-limiting example, differential scanning calorimetry (DSC) provides a method for both assessing the ability of purified lactoferrin to bind metal ions and for assessing the ratio of hololactoferrin to apolactoferrin forms. DSC methods are known to those skilled in the art.

[0093] Dairy Refining The purification method may reduce, minimize or eliminate chemical, enzymatic, acid and / or heat treatment. The purification method may reduce chemical, enzymatic, acid and / or heat treatment. The purification method may minimize chemical, enzymatic, acid and / or heat treatment. The purification method may eliminate chemical, enzymatic, acid and / or heat treatment. The purification method may reduce, minimize or eliminate chemical treatment. The purification method may reduce, minimize or eliminate enzymatic treatment. The purification method may reduce, minimize or eliminate acid treatment. The purification method may reduce, minimize or eliminate heat treatment. The purification method may reduce each of chemical, enzymatic, acid and heat treatment. The purification method may minimize each of chemical, enzymatic, acid and heat treatment. The purification method may eliminate each of chemical, enzymatic, acid and heat treatment. The purification method can remove chemical, enzymatic, and heat treatments.

[0094] Generally, the methods provided herein for the production of purified lactoferrin do not include heat treatments (e.g., pasteurization) typical of industrial purification methods that may destroy (e.g., denature) the native conformational state (with reference to the conformational state found in the natural milk source used to purify lactoferrin). Typical heat treatments used in industrial purification methods may be at about 63°C or higher. Heat treatments may be at 50°C or higher, 51°C or higher, 52°C or higher, 53°C or higher, 54°C or higher, or 55°C or higher. Heat treatments may be at 70°C or higher, 75°C or higher, 80°C or higher, 85°C or higher, 90°C or higher, 95°C or higher, or 100°C or higher. The purification methods may include carrying out processing at temperatures that reduce, minimize, or eliminate the destruction of the native conformational state (with reference to the conformational state found in the natural milk source used to purify lactoferrin) compared to heat treatments typical in industrial purification methods. The purification method may include carrying out processing at a temperature that reduces, minimizes, or eliminates the reduction in lactoferrin bioactivity (with reference to the bioactivity found in the natural milk source used for lactoferrin purification) compared to heat treatments typical in industrial purification methods. The purification method may include receiving the natural milk source at refrigerated temperatures (e.g., temperatures below 15°C, within a temperature range of 2-15°C, etc.). The purification methods described herein may include heat treatment below 50°C. For example, the milk source may be warmed (e.g., to a temperature above 37°C, but not above 55°C) during one or more purification steps. This warming may include temperatures above 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C. This warming may include temperatures above 37°C and below 55°C. The warming may include temperatures above 40° C. and below 55° C. The warming may include temperatures above 45° C. and below 55° C. The warming may include temperatures above 50° C. and below 55° C. The warming may include temperatures above 37° C. and temperatures below 50° C., below 51° C., below 52° C., below 53° C., below 54° C., or below 55° C.The warming may include temperatures above 40° C. and below 50° C., 51° C., 52° C., 53° C., 54° C., or 55° C. The warming may include temperatures above 45° C. and below 50° C., 51° C., 52° C., 53° C., 54° C., or 55° C. The purification methods described herein may include heat treatment below 50° C., 51° C., 52° C., 53° C., 54° C., or 55° C. The purification methods described herein may include maintaining the temperature during purification below 50° C., 51° C., 52° C., 53° C., 54° C., or 55° C. The maintaining of the temperature during purification may include maintaining the temperature throughout the purification. The maintaining of the temperature during purification may include maintaining the temperature at one or more individual steps of the purification method (e.g., chromatography, filtration, and / or drying steps). The temperatures maintained can include a variety of temperatures (eg, different temperature ranges) specific to one or more individual steps of the purification method.

[0095] The purification method may include acid treatment. Without wishing to be bound by theory, acid treatment may be used to remove casein. An example of this is treating the natural milk source or natural milk derivative at a pH that can make casein insoluble in solution prior to lactoferrin purification. Generally, the acid treatments provided herein for the production of purified lactoferrin do not include acid treatments that destroy the native conformational state (e.g., denaturing lactoferrin) and / or reduce the biological activity of lactoferrin (referring to the conformational state or biological activity found in the natural milk source used to purify lactoferrin, respectively). The purification method may include acid treatment at pH 4.0 or higher. The purification method may include acid treatment at pH 3.0 or higher.

[0096] The purification method may include chromatography. The purification method may include ion exchange chromatography. Chromatographic methods such as ion exchange chromatography are known to those skilled in the art. The purification method described herein generally includes cation exchange chromatography. In addition to cation exchange chromatography, the purification method may include ion exchange chromatography steps (such as both cation exchange and anion exchange chromatography) in any order and / or separated by one or more additional purification methods. Resins and matrices for ion exchange chromatography are known in the art. For example, cation exchange resins include, but are not limited to, polymethacrylate and agarose matrices. The purification method may include high pressure liquid chromatography (HPLC).

[0097] Generally, chromatographic methods include one or more equilibration and / or regeneration steps. Illustrative non-limiting examples of equilibration and regeneration steps include (1) a reverse osmosis water rinse, (2) a chemically pure 1 M NaCl rinse, and (3) another reverse osmosis water rinse.

[0098] Generally, chromatographic methods include a loading step. Generally, the loading amount is based on a predetermined binding capacity of the resin and an estimate of the native lactoferrin content of the raw milk feedstock.

[0099] Generally, chromatography method includes one or more elution steps, for example, elution of purified lactoferrin from resin / column in ion exchange chromatography.Elution methods are known to those skilled in the art.Elution methods can include two or more elution steps.Without wishing to be bound by theory, multiple elution steps can be used to first elute contaminants (for example, any other product other than lactoferrin), and then elute desired product (for example, lactoferrin).

[0100] The elution method may include two or more elution steps at different salt concentrations. Without wishing to be bound by theory, one or more early elution steps (e.g., an elution step prior to the elution step containing the desired purified lactoferrin) may be performed to remove undesired proteins and other contaminants.

[0101] The elution method may include a first elution step with a 0.2-0.7M chemically pure NaCl solution. Generally, the first elution step is performed with a 0.2-0.7M chemically pure NaCl solution to remove undesired proteins and other contaminants. Without wishing to be bound by theory, the completion of the first elution may be monitored by UV-Vis spectroscopy and / or colorimetry to monitor for the presence of contaminants such as lactoperoxidase and other enzymes inherent to the raw milk feedstock. For example, the first elution step with a 0.2-0.7M chemically pure NaCl solution may be performed until a lack of protein eluting from the resin is detected by UV-Vis spectroscopy.

[0102] The elution method may include a first elution step of a 0.2M chemically pure NaCl solution. The elution method may include a first elution step of a 0.25M chemically pure NaCl solution. The elution method may include a first elution step of a 0.30M chemically pure NaCl solution. The elution method may include a first elution step of a 0.35M chemically pure NaCl solution. The elution method may include a first elution step of a 0.40M chemically pure NaCl solution. The elution method may include a first elution step of a 0.45M chemically pure NaCl solution. The elution method may include a first elution step of a 0.50M chemically pure NaCl solution. The elution method may include a first elution step of a 0.55M chemically pure NaCl solution. The elution method may include a first elution step of a 0.6M chemically pure NaCl solution. The elution method may include a first elution step of a 0.65 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.7 M chemically pure NaCl solution. The elution method may include a first elution step of a less than 1 M chemically pure NaCl solution.

[0103] The elution method may include a second elution step of 1M chemically pure NaCl solution. In general, lactoferrin is eluted from the resin by elution with 1M chemically pure NaCl solution. The elution method may include a second elution step of about 1M chemically pure NaCl solution.

[0104] The elution method may include a first elution step of a 0.2-0.7M chemically pure NaCl solution and a second elution step of a 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.2M chemically pure NaCl solution and a second elution step of a 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.25M chemically pure NaCl solution and a second elution step of a 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.3M chemically pure NaCl solution and a second elution step of a 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.35M chemically pure NaCl solution and a second elution step of a 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.40M chemically pure NaCl solution and a second elution step of a 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.45M chemically pure NaCl solution and a second elution step of a 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.50M chemically pure NaCl solution and a second elution step of a 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.55M chemically pure NaCl solution and a second elution step of a 1M chemically pure NaCl solution. The elution method may include a first elution step of a less than 1M chemically pure NaCl solution and a second elution step of a 1M chemically pure NaCl solution.

[0105] The elution method may include a first elution step of a 0.25-0.7M chemically pure NaCl solution and a second elution step of about 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.20M chemically pure NaCl solution and a second elution step of about 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.25M chemically pure NaCl solution and a second elution step of about 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.30M chemically pure NaCl solution and a second elution step of about 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.35M chemically pure NaCl solution and a second elution step of about 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.40M chemically pure NaCl solution and a second elution step of about 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.45M chemically pure NaCl solution and a second elution step of about 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.50M chemically pure NaCl solution and a second elution step of about 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.55M chemically pure NaCl solution and a second elution step of about 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.60M chemically pure NaCl solution and a second elution step of about 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.65 M chemically pure NaCl solution and a second elution step of about 1 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.70 M chemically pure NaCl solution and a second elution step of about 1 M chemically pure NaCl solution.

[0106] The elution method may include two or more elution steps at different pH levels. Elution gradients. The elution method may include two or more elution steps at different salt concentrations, different pH levels, and combinations thereof. The elution method may include an elution gradient. The elution method may include a salt elution gradient. The elution method may include a pH elution gradient. The elution method may include both salt and pH elution gradients.

[0107] For each of the ion exchange chromatography steps described above (e.g., equilibration, regeneration, loading, and / or elution), one of skill in the art will recognize appropriate fluid velocities as dependent, for example, on the choice of resin, equipment, milk feed material, etc.

[0108] The purification method may include filtration. Methods of filtration are known to those skilled in the art. The purification method may include microfiltration (usually referring to filtration using a membrane pore size of 0.1-10 μm). The microfiltration may include a membrane pore size of 1-10 μm. The microfiltration may include a membrane pore size of 10 μm. The microfiltration may include a membrane pore size of 1-10 μm. The microfiltration may include a membrane pore size of 0.1-1 μm. The microfiltration may include a membrane pore size of 0.1 μm. The microfiltration may include a membrane pore size of 1 μm. The microfiltration may include a membrane pore size of 0.1, 0.2, 0.3, 0.3, 0.5, 0.6, 0.7, 0.8, 0.9, and / or 1 μm. The microfiltration may include a ceramic filter material.

[0109] Purification methods may include ultrafiltration (usually referring to filtration using membrane pore sizes of 0.01-0.1 μm). Ultrafiltration systems may also be referred to by the molecular weight cut-off size designed for size-based separation between permeate and retentate. Ultrafiltration systems may include 5-30 kDa systems. Ultrafiltration systems may include 5 kDa systems. Ultrafiltration systems may include 10 kDa systems. Ultrafiltration systems may include 15 kDa systems. Ultrafiltration systems may include 20 kDa systems. Ultrafiltration systems may include 25 kDa systems. Ultrafiltration systems may include 30 kDa systems.

[0110] The purification method can include both microfiltration and ultrafiltration, in any order and / or separated by one or more additional purification methods. The purification method can include multiple microfiltration and / or ultrafiltration steps, in any order and / or separated by one or more additional purification methods. As an illustrative, non-limiting example, a first pre-filtration microfiltration step (e.g., using 10 μm filtration media) can be used prior to ion exchange chromatography, and a second microfiltration step (e.g., using 0.1-1.4 μm filtration media) can be used after ion exchange chromatography, followed by an ultrafiltration step (e.g., 5-30 kDa).

[0111] The purification method may include a combination of chromatography and filtration, in any order and / or separated by one or more additional purification methods. The purification method may include a combination of multiple chromatography steps and / or filtration steps, in any order and / or separated by one or more additional purification methods. The purification method may include a combination of microfiltration, ultrafiltration, and ion exchange chromatography, in any order and / or separated by one or more additional purification methods. In an illustrative, non-limiting example, the purification method may include ion exchange chromatography (including elution), followed by microfiltration, then ultrafiltration. In another illustrative, non-limiting example, the purification method may include microfiltration, followed by ion exchange chromatography (including elution), then followed by additional microfiltration, then ultrafiltration.

[0112] Purification methods can include separation of raw dairy products into dairy derivatives, such as separating a natural dairy source into skim milk and cream. For example, raw dairy products can be separated into dairy derivatives prior to chromatography and / or filtration. Methods of separating raw dairy products into dairy derivatives are known to those of skill in the art and include, but are not limited to, cold bowl separation.

[0113] After lactoferrin is purified, the purified product can be dried. Generally, the drying methods provided do not include treatments that destroy the native conformational state (e.g., denaturing lactoferrin) and / or reduce the biological activity of lactoferrin (referring to the conformational state or biological activity found in the native milk source used to purify lactoferrin, respectively). Methods of drying lactoferrin are known to those skilled in the art and include, but are not limited to, freeze-drying / lyophilization, fluidized bed drying, and / or low-temperature spray drying.

[0114] Purity Assessment Natural milk sources, including cow's milk, usually contain several protein components in addition to lactoferrin, examples of which include, but are not limited to, lactoperoxidase, lysozyme, casein, immunoglobulin, lactalbumin, lactoglobulin. Natural milk sources may also contain other components, such as fat and endotoxin. Generally, the purification methods provided herein reduce, minimize, or eliminate components other than lactoferrin. Without wishing to be bound by theory, removing one or more additional components may improve the biological activity and / or safety of lactoferrin.

[0115] Provided herein are lactoferrin compositions that contain an increased percentage of lactoferrin by mass compared to the proportion present in intact lactoferrin-containing dairy products.

[0116] The proportion of lactoferrin can be assessed by mass spectrometry, examples of which include, but are not limited to, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry and / or linear trap quadrupole Orbitrap Velos mass spectrometry. Generally, the mass spectrometry assessment involves quantifying the area under the peak corresponding to lactoferrin and the area under the peak not corresponding to lactoferrin. In some cases, lactoferrin can be associated with multiple peaks, such as ionization peaks corresponding to lactoferrin. The peaks corresponding to lactoferrin can include peaks corresponding to full-length post-translationally modified (e.g., glycosylated) lactoferrin. The peaks corresponding to full-length post-translationally modified lactoferrin are generally in the range of about 80,000-86,000 m / z. The exact peaks of the post-translationally modified full-length may vary, such as to reflect different glycosylation states. In some cases, a peak having an m / z of 79,000-90,000 (inclusive) can be considered to correspond to lactoferrin. The area under the peaks not corresponding to lactoferrin includes all other peaks, with the potential exception of the ionization peak associated with lactoferrin at about 41,500 m / z (e.g., with m / z between 41000 and 42000). The area under the peaks not corresponding to lactoferrin may include peaks with m / z between 18000 and 80000 (excluding peaks with m / z between 41000 and 42000). It is also possible to make specific comparisons between lactoferrin and peaks corresponding to lactoperoxidase (e.g., with m / z between 77000 and 78000). The area under the peaks not corresponding to lactoferrin may include peaks with m / z between 18000 and 45000 (excluding peaks with m / z between 41000 and 42000). Linear trap quadrupole Orbitrap Velos mass spectrometry also allows for the quantification of the proportion of lactoferrin relative to other components in a sample. For example, quadrupole Orbitrap Velos mass spectrometry can quantify the proportion of lactoferrin by determining the proportion of peptide spectral matches (PSMs).

[0117] The percentage of lactoferrin can be assessed by liquid chromatography, such as high performance liquid chromatography (HPLC), which can include quantifying the area under the peak corresponding to lactoferrin and the area under the peak not corresponding to lactoferrin.

[0118] In assessments involving quantification of the peaks, the area under one or more peaks not corresponding to lactoferrin present in the raw lactoferrin-containing dairy product may be below the detection limit of the purified lactoferrin composition. In some cases, each area under a peak not corresponding to lactoferrin may be below the detection limit.

[0119] The proportion of lactoferrin can be assessed by enzyme-linked immunosorbent assay (ELISA). In some cases, the ELISA can distinguish the proportion of lactoferrin in the native protein conformation, such as by using an antibody that specifically binds to the native protein conformation of lactoferrin.

[0120] In purifying lactoferrin, a particular component that is typically present in raw milk sources and that is reduced, minimized, or eliminated is lactoperoxidase, which is frequently present in detectable amounts in purified lactoferrin compositions produced by typical industrial processes.

[0121] Provided herein are lactoferrin compositions, wherein at least 70% of the composition is purified lactoferrin. The compositions include those in which at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the composition is purified lactoferrin. The compositions include those in which at least 75% of the composition is purified lactoferrin. The compositions include those in which at least 80% of the composition is purified lactoferrin. The compositions include those in which at least 85% of the composition is purified lactoferrin. The compositions include those in which at least 90% of the composition is purified lactoferrin. The compositions include those in which at least 95% of the composition is purified lactoferrin. The compositions include those in which at least about 100% of the composition is purified lactoferrin. The compositions include those in which at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the composition is purified lactoferrin. Compositions include those in which at least 91% of the composition is purified lactoferrin. Compositions include those in which at least 92% of the composition is purified lactoferrin. Compositions include those in which at least 93% of the composition is purified lactoferrin. Compositions include those in which at least 94% of the composition is purified lactoferrin. Compositions include those in which at least 95% of the composition is purified lactoferrin. Compositions include those in which at least 96% of the composition is purified lactoferrin. Compositions include those in which at least 97% of the composition is purified lactoferrin. Compositions include those in which at least 98% of the composition is purified lactoferrin. Compositions include those in which at least 99% of the composition is purified lactoferrin.

[0122] Provided herein is a lactoferrin composition that has an increased ratio of lactoferrin:lactoperoxidase compared to the ratio in unprocessed lactoferrin-containing dairy products.The ratio of lactoferrin:lactoperoxidase can be evaluated according to methods known to those skilled in the art, such as the purity evaluation methods described herein (e.g., mass spectrometry, HPLC, and / or ELISA).For example, evaluation can include quantifying the area under the peak(s) corresponding to lactoferrin and the area under the peak corresponding to lactoperoxidase.

[0123] The endotoxin present or possibly present in natural milk source can be reduced, minimized or eliminated.Without wishing to be bound by theory, the removal of endotoxin can improve the safety of purified lactoferrin composition.Methods for evaluating endotoxin level are known to those skilled in the art.

[0124] Also provided herein are methods for assessing the purity of a purified lactoferrin composition, such as using any of the purity assessment methods described herein.

[0125] Pharmaceutical Compositions Provided herein are pharmaceutical compositions comprising any one of the purified lactoferrin compositions described herein and one or more pharma- ceutically acceptable excipients.

[0126] As used herein, "pharmaceutical composition" is meant to encompass compositions suitable for administration to a subject, such as a mammal, particularly a human. Generally, a "pharmaceutical composition" is sterile and preferably free of contaminants that may induce an undesirable response in a subject (e.g., the compound(s) in the pharmaceutical composition are pharmaceutical grade). The pharmaceutical composition can be designed for administration to a subject or patient in need of administration via many different routes of administration, including, but not limited to, topical routes such as dermal (e.g., wounds), mucosal, respiratory, oral (including alimentary tract), and nasal formulations.

[0127] "Pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" refer to excipients, diluents, carriers, and adjuvants that are useful in preparing pharmaceutical compositions that are generally safe, non-toxic, and not biologically or otherwise undesirable, and include excipients, diluents, carriers, and adjuvants that are acceptable for veterinary as well as human pharmaceutical use. As used in the specification and claims, "pharmaceutically acceptable excipients, diluents, carriers, and adjuvants" includes both one and more of such excipients, diluents, carriers, and adjuvants.

[0128] In pharmaceutical applications, lactoferrin may be incorporated into oral dissolving formulations. This may include, but is not limited to, oral dissolving tablets, lozenges, gums, oral thin films, or direct application of lactoferrin in excipient powders. Pharmaceutically acceptable excipients include, but are not limited to, sodium steryl humate, citric acid, xylitol (Xylisorb XTAB 240), sorbitol (Neosorb XTAB 200S or Neosorb P60 W), mannitol, maltitol (Sweetpearl P300 DC), and / or other sugar alcohols, including any combination thereof. Without wishing to be bound by theory, oral dissolving formulations may promote retention of lactoferrin in the oral cavity and nasopharynx for local targeting of lactoferrin. Oral dissolving formulations may be prepared using techniques known in the art, examples of which include fluidized bed drying, or any other drying technique combined with compressing the product into a final dosage form. The oral formulation may include a bioadhesive. Applicable bioadhesives are known to those skilled in the art, examples of which include those described in Duan et al. (Applications of Bioadhesives: A Mini Review; Front Bioeng Biotechnol. 2021;9: 716035.), which is incorporated herein by reference for all purposes. The oral formulation may include an astringent. Applicable astringents are known to those skilled in the art, examples of which include, but are not limited to, alum, acacia, sage, yarrow, witch hazel, bayberry, distilled vinegar, persimmon, green tea, black tea, citric acid, cranberry juice and / or extract, grapefruit juice and / or extract, or inorganic acid.

[0129] Oral formulations may be particularly applicable for gastrointestinal applications. Oral formulations including gastrointestinal applications may include microencapsulation of lactoferrin. Oral formulations including gastrointestinal applications may include microencapsulation of pharmaceutical compositions including any one of the purified lactoferrin compositions described herein, including enteric coated formulations. Oral formulations including gastrointestinal applications may include microencapsulation of pharmaceutical compositions including any one of the purified lactoferrin compositions in combination with one or more additional active agents (e.g., enteric coated multi-component formulations). Microencapsulation formats are known to those skilled in the art, examples of which include those described in Singh et al. (Microencapsulation: A promising technique for controlled drug delivery; Res Pharm Sci. 2010 Jul-Dec;5(2): 65-77.), which is incorporated herein by reference for all purposes.

[0130] For medical applications targeting wound healing, lactoferrin may be incorporated into a formulation that promotes contact of lactoferrin with the wound. A wound may refer to any injury to the tissue of a subject. Injuries include, but are not limited to, burns (e.g., fire / heat, chemicals, electricity, radiation (e.g., ultraviolet light or radioactive materials), friction, cuts (e.g., punctures, lacerations, abrasions, incisions (e.g., surgical incisions), amputations, infections, and ulcers). A wound may include an open wound. A wound may include an ulcer, such as a venous or arterial ulcer. Multiple forms of injury may exist together. For example, as an illustrative, non-limiting example, an injury may include both a burn with an infection, or a cut with an infection. A tissue may include, but is not limited to, skin tissue and lung tissue. Injuries to multiple tissues may exist together. For example, as an illustrative, non-limiting example, an injury may include injury to both skin tissue and lung tissue. A formulation that promotes contact of lactoferrin with the wound may include a bioadhesive. Applicable bioadhesives are known to those skilled in the art, examples of which include those described herein. Delivery formats of the wound healing formulations include, but are not limited to, gel foams, hydrogels, injected dressings, powders, sprays, and inhalation formats (e.g., nebulizers, inhalers, and mist formats).

[0131] For pharmaceutical applications directed to promoting antibacterial applications, lactoferrin may be incorporated into formulations that promote the antibacterial activity of lactoferrin. Antibacterial use may include treatment of active infection. Antibacterial use may include prevention and / or treatment of infection. For example, prophylactic prevention and / or treatment of infection may include application to wounds before infection results.

[0132] The formulation and / or delivery format can promote multiple uses and / or properties of lactoferrin. As a non-limiting illustrative example, a formulation can promote both the wound healing and antimicrobial properties of lactoferrin, such as a formulation or delivery format that can both promote cell growth and prevent microbial biofilm formation.

[0133] Lactoferrin may also be incorporated into formulations called "medical foods" which are usually considered to be foods intended for the administration of these products. These applications include incorporation into gums or other chewable products for slow release of lactoferrin. Medical food formulations such as gums may contain excipients such as xylitol, powdered sugar, gum base, sorbitol, mannitol, sucralose, and / or corn syrup, including any combination thereof. In the case of gums, lactoferrin may be incorporated either throughout the gum or as an outer coating of the gum. Medical food formulations may be prepared using techniques known in the art, examples of which include fluidized bed drying or any other drying technique, and final preparation with gum base to form the final dosage form.

[0134] The formulation may include sodium steryl humate, carbomer, sodium hydroxide, glycerin, sodium benzoate, benzoic acid, citric acid, and / or combinations thereof. The formulation may include xylitol, sorbitol, mannitol, maltitol, sugar alcohols, sucralose, and / or combinations thereof. The formulation may include isomalt, microcrystalline cellulose, sodium carboxymethylcellulose, and / or combinations thereof. The formulation may include powdered sugar, gum base, corn syrup, gum arabic, and / or combinations thereof. The formulation may include HiG PWD-04, encapsulated powdered flavor, anhydrous or natural caffeine, encapsulated sucralose or sucralose, encapsulated acesulfame-K and aspartame, aspartame, acesulfame-K, triacetin, silicon dioxide, and / or combinations thereof.

[0135] The formulation can include a coagulant, examples of which include, but are not limited to, thrombin, amylopectin, kaolin, and / or combinations thereof.

[0136] Pharmaceutical compositions, such as orally soluble formulations and medical foods, may have an optimized pH to maximize protein stability and activity. The mode of administration, including the use of selected excipients, may contribute to improving the bioavailability, stability, and / or biological activity of the protein.

[0137] Also provided herein are compositions that can take any of the forms of pharmaceutical compositions described herein, but that are not pharmaceutical in nature, such as cosmetics (e.g., skin or oral beauty products).

[0138] Treatment and risk prevention / reduction Provided herein is a method for treating a disease or condition by administering a therapeutically effective amount of any of the pharmaceutical compositions described herein. Examples of the pharmaceutical composition include any one of the pharmaceutical compositions formulated for delivery to the oral cavity, nasal cavity, or other tissues (e.g., skin tissue or lung tissue). Diseases or conditions include, but are not limited to, pathogenic diseases, gastrointestinal (GI) infections, wound infections, allergies, and inflammatory conditions.

[0139] Also provided herein are methods of modulating immune responses (e.g., promoting anti-inflammatory activity) by administering a therapeutically effective amount of any one of the purified lactoferrin compositions described herein, including administering any of the pharmaceutical compositions described herein. The term "modulate" includes maintaining biological activity, inhibiting biological activity (partially or completely), and stimulating / activating biological activity (partially or completely). The term also includes decreasing or increasing (e.g., enhancing) biological activity. For example, administering a therapeutically effective amount of purified lactoferrin can reduce inflammation by promoting anti-inflammatory activity (e.g., in the context of an inflammatory disease).

[0140] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect, examples of which include prevention, risk reduction, amelioration and / or resolution of an infectious disease, such as a viral, fungal (e.g., yeast), or bacterial infection. Treatment may be prophylactic in terms of completely or partially preventing the disease or symptoms, and / or may be therapeutic in terms of partially or completely curing the disease and / or side effects caused by the disease. Treatment encompasses any treatment of a mammalian, particularly a human, disease, including (a) prophylactically treating (completely or partially preventing) the development of a disease or symptoms of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed as having the disease (e.g., as in the case of a subject at risk for an infectious disease), (b) suppressing the disease (e.g., eliminating the infection and / or reducing the infection below detectable limits), and (c) alleviating the disease (e.g., reducing the microbial load associated with the infectious disease).

[0141] Also provided herein is a method for reducing the risk of pathogenic disease, comprising administering any of the pharmaceutical compositions or compositions described herein to a subject. For example, reducing the risk of pathogenic disease can include, but is not limited to, administering a pharmaceutical composition or composition described herein to a wound at risk of pathogenic disease (e.g., infection by a pathogen). A subject at risk of pathogenic disease can be exposed to a pathogen. A subject at risk of pathogenic disease can be diagnosed with infection by a pathogen. Reducing the risk can include administering a pharmaceutical composition or composition described herein that reduces the risk of pathogenic disease compared to a subject not administered the pharmaceutical composition or composition.

[0142] Also provided herein are methods for treating a subject identified as being at risk for a pathogenic disease, the methods comprising administering to the subject any of the pharmaceutical compositions or compositions described herein, thereby reducing the risk of the pathogenic disease.

[0143] "Therapeutically effective amount" or "effective amount" means the amount of a compound that, when administered to a mammal or other subject for treating a disease, condition, or disorder, is sufficient to effect such treatment for the disease, condition, or disorder. A "therapeutically effective amount" will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0144] The subject compound can be administered to the subject alone or in combination with an additional active agent. The terms "agent", "compound" and "medicine" are used interchangeably herein. The method can further include co-administering a second agent, either in combination or sequentially, examples of which include small molecules, antibodies, antibody fragments, antibody drug conjugates, aptamers, proteins, antibiotics, antiviral agents, antibacterial agents, antimicrobial agents, antifungal agents, and / or vaccines. In some embodiments, the method further includes administering radiation therapy to the subject.

[0145] The terms "co-administration" and "in combination with" include administration of two or more therapeutic agents simultaneously, together, or sequentially without specific time limitations. In one embodiment, the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effects simultaneously. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. The term "unit dosage form" as used herein refers to a physically discrete unit suitable as a unitary dose for human and animal subjects, each unit containing a predetermined amount of a compound (e.g., an aminopyrimidine compound described herein) calculated to be sufficient to produce a desired effect, together with a pharma- ceutically acceptable diluent, carrier, or excipient. The specifications of the unit dosage form depend on the particular compound employed, the effect to be achieved, and the pharmacodynamics associated with each compound in the host. In certain cases, the combination provides an improved effect compared to either component alone. In some cases, the combination provides a superadditive or synergistic effect compared to the combined or additive effect of the components. For multiple doses, the two agents may be alternating directly, or, for example, two or more doses of one agent may be alternating with a single dose of the other agent.

[0146] Also provided herein is a method of treating a pathogenic disease. The method can include administering a pharmaceutical composition to a subject, examples of which include any one of the pharmaceutical compositions described herein (e.g., any one of the pharmaceutical compositions formulated for delivery to the oral or nasal cavity). The administration can be prophylactic. The subject can be at risk of exposure to a pathogen. For example, the risk of exposure to a pathogen can include, but is not limited to, administration to a wound at risk of infection by the pathogen. The subject can be exposed to a pathogen. The subject can be diagnosed with an infection by a pathogen.

[0147] Pathogens include, but are not limited to, microorganisms transmitted orally and / or nasally. Pathogens include, but are not limited to, airborne microorganisms. Pathogens include, but are not limited to, viruses, such as coronaviruses (e.g., SARS-CoV-2). Pathogens include, but are not limited to, bacteria and / or fungi (e.g., yeast).

[0148] Also provided herein are methods for promoting wound healing (e.g., through promoting anti-inflammatory activity, promoting cell proliferation, and / or preventing infection) by administering a therapeutically effective amount of any one of the purified lactoferrin compositions described herein, including administering any of the pharmaceutical compositions described herein.

[0149] Manufacturing method Provided herein is any one of the methods of purified lactoferrin composition described herein, including any one of the pharmaceutical compositions described herein.Method includes any one of the purification steps described in the "Dairy Purification" section, including the combination of steps described therein.Illustrative non-limiting examples are described in the Examples section herein. EXAMPLES

[0150] Below are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure the accuracy of the numerical values ​​used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should naturally be allowed for.

[0151] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, within the skill of those in the art. Such techniques are explained fully in the literature, e.g., TE Creighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993);AL Lehninger, Biochemistry (Worth Publishers, Inc., current addition);Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989);Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.);Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990);Carey and Sundberg Advanced Organic Chemistry 3rd Edition (East, Pennsylvania: Mack Publishing Company, 1992); rd Ed. (Plenum Press) Vols A and B (1992).

[0152] Example 1. Purification of lactoferrin Lactoferrin was purified as shown in Figure 1. Briefly, raw milk (e.g., unprocessed milk that has not been chemically, enzymatically, acidically, or thermally treated prior to the purification of lactoferrin) is (1) diverted before entering the industry-standard milk processing workflow, (2) run through an ion exchange resin / column, the effluent is typically returned to the standard milk processing workflow, the lactoferrin-containing eluate is collected, (3) the collected eluate is filtered, and (4) the purified lactoferrin is processed. Notably, all other known lactoferrin purification methods start from a milk source that is obtained after the industry-standard milk processing workflow, which typically includes low-temperature heat pasteurization. Furthermore, because natural unprocessed dairy products (e.g., raw milk) were used as the raw material, lactoferrin contained the appropriate post-translational modifications that may be involved in biological activity, in contrast to recombinantly produced lactoferrin.

[0153] Bovine lactoferrin was isolated and purified using a chromatography column to retain native post-translational modifications, glycosylation, and bound iron. The column was packed with a selected cation exchange resin (either polymethacrylate or agarose matrix) and equilibrated and regenerated by (1) rinsing with reverse osmosis water, (2) rinsing with chemically pure 1 M NaCl, and (3) rinsing again with reverse osmosis water.

[0154] Raw, unprocessed, unprocessed whole bovine milk (<24 h after milking) was obtained directly from dairy shipping bins or silos prior to separation, skimming, heating, and / or pasteurization. Lactoferrin was purified from both raw colostrum (RC) and raw whole milk (RM).

[0155] The milk was filtered through a 10 μm filter media to remove large particulate matter, warmed to above 37°C, maintained at a temperature below 63°C (generally considered the starting temperature for pasteurization), and loaded onto the chromatography column. The loading volume was based on a pre-determined binding capacity and an estimate of the native lactoferrin content of the raw milk feed. During the loading process, all flow-through was returned to the pasteurizer balance tank, returning the plant feed to a point prior to separation and pasteurization.

[0156] Once loading was complete, the resin was rinsed with reverse osmosis water to remove any remaining milk compounds that were not bound to the resin. The first elution involved washing the resin with a chemically pure NaCl solution (0.2-0.7M) and was monitored and assessed for completion (i.e., lack of protein eluting from the resin) by UV-Vis spectroscopy and colorimetry, which monitored for the presence of contaminants such as lactoperoxidase and other enzymes inherent to the raw milk feedstock. In particular, the first NaCl was run until the elution fractions were free of lactoperoxidase, which is generally the major contaminant, as assessed by a peroxidase colorimetric assay using a colorimetric peroxidase substrate. The fractions from the first elution were retained in a segregated container. The resin was then rinsed with reverse osmosis water. A second elution was run with 1M NaCl to remove the isolated lactoferrin. The second elution fraction was retained in a segregated container and then (1) filtered through a ceramic microfiltration filter material (0.1-1.4 μm) and (2) concentrated using an ultrafiltration system (5-30 kDa).

[0157] The desalted eluate was then freeze-dried, but may also be dried directly into a fluid bed dryer over pharmaceutical grade excipients. Purified lactoferrin produced according to the methods described herein is called hyacinth lactoferrin ("hyacinth"), lactoferrin (only those not accompanied by the phrases "laboratory grade" or "commercial supplement grade"), ODT-SC210, and API-E2. Various names may refer to purified lactoferrin produced according to variations of the methods described herein.

[0158] Example 2. Evaluation of lactoferrin purity The purity of the purified lactoferrin produced according to Example 1 was evaluated.

[0159] Typically, SDS-PAGE gels are used to assess the purity of reference grade protein-derived products (including reference grade lactoferrin). However, SDS-PAGE gels are notorious for producing inflated purity results depending on the experimental conditions (Kurien and Scofield Methods Mol Biol. 2012;869: 633-640). Therefore, more sensitive mass spectrometry, HPLC, and ELISA were used to assess purity.

[0160] The purity of lactoferrin was assessed by MALDI-TOF mass spectrometry. Dried samples were dissolved in water at a concentration of 10 mg / mL. The dissolved samples were mixed with an equal volume of saturated sinapinic acid in 50% acetonitrile containing 0.1% trifluoroacetic acid. The sample / matrix mixture (2 μL) was placed on a M1P 384 polished steel MALDI plate. MALDI mass spectra were acquired in positive ion mode at m / z 2001–20162 Da (2–20 kDa), 10039–40026 Da (10–40 kDa), and 19780–100000 Da (20 kDa–100 kDa). The instrument was calibrated using Protein Calibration Standard II (Bruker) for these mass ranges. MS spectra were analyzed using F1exAnalysis 3.4 (Bruker Daltonics, Billerica, MA).

[0161] Purity results assessed by MALDI-TOF (approximately 18 kDa to 100 kDa) for purified lactoferrin according to Example 1 from two different natural raw milk sources (colostrum and whole milk) are shown in Figures 2A and 2B, respectively, and quantified in Tables 1A and 1B. The prominent peak above approximately 80,000 m / z corresponds to glycosylated lactoferrin, and the peak at approximately 41,500 m / z corresponds to the ionization peak of lactoferrin. Notably, the typical contaminating peak associated with lactoperoxidase (approximately 78,000 m / z) was below the detection limit. Quantification of the mass spectrometry profiles revealed that more than 75% of the relative area under the curve (AUC) determined for the quantified peaks in the range of 18 kDa to 100 kDa corresponded to the desired glycosylated lactoferrin peak at 80-85 kDa, and nearly 100% corresponded to lactoferrin when the ionization peak at about 41,500 m / z was included (peaks selected for quantification, in other words peaks considered as true peaks above background noise, were determined by F1exAnalysis 3.4). Thus, the results demonstrate that the purification method described herein produced lactoferrin of high purity from various raw natural dairy products. [Table 1A] [Table 1B]

[0162] The purity of an over-the-counter (OTC) lactoferrin supplement [Jarrow Formulas] (FIG. 3A) and a lactoferrin source advertised as a laboratory reagent grade product [Sigma Bovine Colostrum Lactoferrin] (FIGS. 3B and 3C) was also assessed by MALDI-TOF (approximately 18 kDa to 100 kDa) as described above and quantified in Tables 2A, 2B, and 2C, respectively. In contrast to lactoferrin produced according to Example 1, the mass spectrometry profiles reveal contaminating peaks (particularly lactoperoxidase) for the OTC supplement and both laboratory reagent grade lactoferrin sources (see peak at 77806 m / z in FIG. 3B). Quantification of the mass spectrometry profiles revealed that only 25.3%, 52.3%, and 11.9% of the relative AUC determined for the quantified peaks in the 18-100 kDa range corresponded to the desired glycosylated lactoferrin peak at 80-85 kDa, respectively. Even when considering the lactoferrin ionization peak at approximately 41,500 m / z, only 33.5%, 66.4%, and 15.9% of the combined relative AUC corresponded to lactoferrin, respectively. Thus, the results demonstrate that the purification method described herein produced lactoferrin of higher purity than available OTC supplement and laboratory reagent grade lactoferrin sources. [Table 2A] [Table 2B] [Table 2C]

[0163] The purity of lactoferrin was also evaluated by linear trap quadrupole Orbitrap Velos mass spectrometry. The Orbitrap Velos mass spectrometry profile of lactoferrin produced according to Example 1, shown in Figure 4 and quantified in Table 3A, revealed that more than 80% of the identified peptide spectral matches (PSMs) corresponded to the desired lactoferrin. In contrast, the Orbitrap Velos mass spectrometry profile of the OTC supplement, shown in Table 3B, revealed that only about 62% of the identified PSMs corresponded to lactoferrin. Notably, about 11% of the identified PSMs corresponded to lactoperoxidase, the main contaminant. Therefore, this result demonstrates that the purification method described herein produces high-purity lactoferrin with a purity higher than other available lactoferrin sources. [Table 3A] [Table 3B-1] [Table 3B-2]

[0164] The purity of lactoferrin was also assessed by ELISA. As shown in Figure 5, when loaded with the same amount of protein by weight, lactoferrin produced according to Example 1 ("Hyacinth") demonstrated a 30% increase in antibody binding compared to the existing research standard. Thus, this data demonstrated that Hyacinth protein purified protein (API-E2) achieved higher purity and / or retained a greater fraction of the native conformational protein state compared to other research grade reference products.

[0165] The purity of lactoferrin was assessed by HPLC using a Thermo Fisher U3000 protein purification system with a Tricorn5 / 150 column packed with Cytiva BigBeads using a NaCl gradient. As shown in Figure 5B, API-E2 processed by HPLC demonstrated a single peak corresponding to lactoferrin, indicating approximately 100% purity.

[0166] This data demonstrates that the purification method of Example 1 achieved higher purity lactoferrin compared to existing available reagents, particularly in its ability to reduce lactoperoxidase contamination.

[0167] Example 3. Evaluation of lactoferrin activity through extracellular matrix association Developing an infection requires a multi-step process through which the virus progresses. First, once the virus enters the host, it targets the cell by binding to the host's heparan sulfate (HS) proteoglycans in the extracellular matrix (ECM), facilitating the binding of the virus particle to its specific receptor on the cell surface. The virus is then internalized and replicated within the host cell. Typically, antiviral drugs focus on inhibiting key viral replication proteins, or specific receptors on the cell surface, but the nonspecific cell targeting mechanism of HS in ECM binding may also be inhibited, hindering or preventing the virus from binding to the target cell. Because this route is relatively nonspecific and requires coating the ECM of exposed cells, conventional therapeutics utilizing this approach usually must be administered at relatively high local concentrations for maximum efficacy and generally suffer from the weakness of impurities and / or toxicity. Thus, lactoferrin preparations with higher purity (e.g., pharmaceutical grade standard) would provide a significant reduction in the dosage. Furthermore, when prepared in a manner that preserves native function (e.g., preserves native conformation, post-translational modifications, iron-binding ability, etc.), lactoferrin will retain its effectiveness relative to its activity in raw milk. These preparations (e.g., those prepared in Example 1) allow lactoferrin to be used, stored, formulated, and / or delivered in a greater capacity than other lactoferrin products.

[0168] To evaluate the biological activity of lactoferrin, especially its potential as an antiviral drug, its binding to ECM was evaluated. Purified lactoferrin was produced according to Example 1. Caco-2 cells, a human-derived cell culture, were cultured on a cover slip and allowed to grow for 3 days to allow the cells to proliferate and the ECM to fully develop. Purified lactoferrin was then added to the cells at various concentrations for 2 hours at 37°C, allowing it to bind as expected. To directly observe the localization of the API, immunofluorescence (IF) imaging was used to stain both the API and the cell membrane marker E-cadherin.

[0169] As shown in Figure 6, enrichment of purified lactoferrin in the ECM was observed as the concentration increased. Although primary internal localization was also observed at low concentrations, partial localization of lactoferrin in the intracellular space was expected given that lactoferrin has a known role in cells and receptors that allow its uptake. To quantify API localization, the absolute intensity of the fluorescent signal both just outside / along the E-cadherin mark and inside the cell was measured. Quantification of lactoferrin localization demonstrated enrichment in the ECM as the concentration increased, as shown in Figure 7. This result indicates that purified lactoferrin exhibited bioactivity to bind to the ECM.

[0170] Primary buccal cells were then cultured according to standard protocols (see, e.g., Russo et al. Cytotechnology. 2016 Oct;68(5):2105-2114; incorporated herein by reference for all purposes). For both API-E2 lactoferrin and the commercial laboratory grade standard, lactoferrin was added to a final concentration of 100 μg / mL in the medium for 1 hour at 37° C. This is approximately the concentration at which API-E2 lactoferrin was previously found to begin to saturate binding of cultured cells. Immunofluorescence imaging was performed as previously described. As shown in FIG. 8, API-E2 lactoferrin demonstrated strong binding along the ECM of buccal cells in these conditions (top row). However, the commercial laboratory grade standard demonstrated only limited ECM association of lactoferrin under the same conditions (bottom row). This result strongly suggests that API-E2 lactoferrin is more potent and bioactive with respect to the critical host cell ECM association required for both the antiviral activity of the protein and the biofilm mitigating activity of antibiotics.

[0171] Example 4. SARS-CoV-2 Antiviral Activity A cytopathic effect (CPE) reduction assay was performed to evaluate the antiviral activity of purified lactoferrin against SARS-CoV-2.

[0172] Vero E6 cells were seeded in 96-well cell culture plates at a density of 80-100% confluent cells. The cells were incubated for 2 hours at 37°C with 3-fold serial dilutions of SC210 starting at a concentration of 1 mg / mL. The cells were then mock infected (analysis of compound cytotoxicity) or infected with purified lactoferrin produced according to Example 1, reagent grade lactoferrin, or remdesivir at an MOI of 0.001 in a total volume of 150 μl of medium. On day 3 post-infection, cells were stained with neutral red dye for 2 hours, the dye was extracted with 50:50 Sorensen citrate buffer / ethanol for 30 minutes, and cell viability was assessed by reading the optical density at OD540nm and determining the EC50 (50% effective antiviral concentration). As shown in Figure 9 and quantified in Table 4, purified lactoferrin inhibited viral growth at lower concentrations than the equivalent research grade reference product. The results demonstrated that purified lactoferrin produced according to Example 1 inhibited SARS-CoV-2 viral infection better than reagent grade, suggesting improved biological activity and / or purity. [Table 4]

[0173] Example 5. Evaluation of lactoferrin bioactivity by differential scanning calorimetry Lactoferrin has several important biological activities related to antibacterial activity, including the important activities of iron chelating and host cell binding. Naturally, lactoferrin exists in both iron-bound (hololactoferrin) and iron-free (apolactoferrin) forms. Without wishing to be bound by theory, hololactoferrin should be substantially more stable than apolactoferrin, given that lactoferrin binds strongly to iron.

[0174] Differential scanning calorimetry (DSC) was used to assess the hololactoferrin and apolactoferrin forms by monitoring the unfolding temperature of lactoferrin samples, using a nanoDSC (TA Instruments, Lindon, UT) following standard DSC protocols known to those of skill in the art (see, e.g., Hinz et al “MEASUREMENT AND ANALYSIS OF RESULTSOBTAINED ON BIOLOGICAL SUBSTANCES WITH DIFFERENTIAL SCANNING CALORIMETRY”; which is incorporated herein by reference for all purposes).

[0175] API-E2 lactoferrin (10 mg / mL) produced as described herein was evaluated by DSC. As shown in FIG. 10 (solid line), API-E2 lactoferrin showed two peaks, from the left, corresponding to apolactoferrin as the major peak and hololactoferrin as the minor peak. The absence of other peaks indicates the absence of contaminants and impurities, allowing API-E2 to be evaluated as approximately 100%. This result indicates that the presence of iron stabilizes lactoferrin, resulting in a shift to a higher denaturation temperature in the DSC assay. Thus, the DSC assay demonstrated the ability to distinguish between apolactoferrin and hololactoferrin forms in a given sample. Furthermore, the absence of other peaks also indicates the purity of API-E2 lactoferrin. The API-E2 apolactoferrin peak has a Tm of 60.2±0.8° C. and a delta H of 466.1±9.2 kJ / mol. The API-E2 hololactoferrin peak has a Tm of 88.38±0.8°C and a delta H of 632.7±7.2 kJ / mol. Hololactoferrin naturally constituted an average of 8.74±1.39% of the total API-E2 lactoferrin, with apolactoferrin making up the remainder of the samples tested. However, the percentage of hololactoferrin can of course vary. By this technique, it was calculated that this API-E2 contained essentially 100% pure lactoferrin. [Table 5]

[0176] This assay was also performed on heat-treated API-E2 lactoferrin. API-E2 lactoferrin was heated over the range of 50-100 °C. Notably, the heat range used in the DSC assay is comparable to the temperatures typically used in industry prior to purification of lactoferrin (e.g., above 63 °C). As shown in Figure 10 (dashed line), heat-treated API-E2 lactoferrin showed a loss of both detectable peaks, indicating that both apolactoferrin and hololactoferrin forms were completely denatured after heat treatment.

[0177] DSC assays were further used to assess the bioactivity of lactoferrin, specifically the iron binding capacity of API-E2 lactoferrin. Iron chloride (FeCl3) was added to API-E2 lactoferrin at a 1000-fold molar excess. In the presence of excess iron, API-E2 lactoferrin binds iron to its vacant iron binding sites. If API-E2 retains its bioactivity throughout production, all of the apolactoferrin is expected to transition to the more stable hololactoferrin form, as determined by the presence of the corresponding DSC peaks. As shown in Figure 11A, API-E2 lactoferrin, in the absence of excess iron, exhibited two peaks, one corresponding to apolactoferrin as a major peak on the left and one corresponding to hololactoferrin as a minor peak on the right. Upon addition of excess iron, 100% of the available apolactoferrin transitioned to the hololactoferrin form, as indicated by the absence of detectable apolactoferrin peaks and now a single lactoferrin peak (right peak) being observed. This result indicates that iron-binding bioactivity is fully retained in API-E2 lactoferrin produced using the methods described herein.

[0178] Standard laboratory grade lactoferrin and commercial supplement grade lactoferrin were also evaluated by DSC under the conditions used to evaluate API-E2 above. As shown in FIG. 11B, the peaks of standard laboratory grade lactoferrin were significantly smaller and less distinct than those observed for API-E2, and contained one extra peak likely due to an impurity. The curve had a non-specific upward slope, as typically observed in products with highly denatured proteins present in the sample. The results showed that the estimated ratio of apolactoferrin to hololactoferrin peaks was nearly equal, indicating that apolactoferrin was lost during processing. Similarly, both apolactoferrin and hololactoferrin peaks denatured at lower temperatures of 55°C and 85°C, respectively, indicating increased instability due to previous chemical, enzymatic, and / or thermal treatments, resulting in loss of iron-binding capacity. Also shown in FIG. 11B, commercial supplement grade lactoferrin (lower line) showed no detectable peaks. This indicates that the product was either completely denatured or lacked the presence of lactoferrin molecules.

[0179] Example 6. Evaluation of lactoferrin bioactivity against salivary bacteria by pH test Salivary bacteria (including Lactobacillus, Lactococcus, and Streptococcus mutans) produce acid during growth. This acidification is strongly associated with dental caries, especially when it results in a pH below 5.5, which is the clinically relevant threshold. Slurries of these bacteria were treated with sucrose and various concentrations of API-E2 lactoferrin to stimulate growth. Upon addition of sucrose and API-E2 lactoferrin, the pH was adjusted to 7 by titration and the subsequent change in pH was monitored over the next 6 hours.

[0180] As shown in Figure 12, in the presence of sucrose without API-E2 lactoferrin treatment, salivary bacteria produced acid, resulting in a drop in pH below 5.5 within 4 hours. However, in the presence of sucrose at either 1 mg / mL or 10 mg / mL API-E2 lactoferrin, the pH remains above 5.5 throughout the 6 hour test period. This result indicates that the presence of API-E2 reduced bacterial acid production in a concentration-dependent manner, demonstrating the antibacterial bioactivity of API-E2 lactoferrin.

[0181] Example 7. Evaluation of lactoferrin bioactivity following surface treatment of poultry Chicken breasts were inoculated with human salivary bacteria (including Lactobacillus, Lactococcus, Streptococcus mutans, and Porphyromonas gingivalis) in a slurry as described above in the pH test and left at 37°C for 6 days.

[0182] As shown in Figure 13, without treatment, the chicken breasts were covered with bacterial growth at the end of the six day time frame. However, with treatment with 3 mg / mL API-E2 lactoferrin, no detectable growth of any bacterial colonies was observed after six days, demonstrating the antibacterial bioactivity of API-E2 lactoferrin after surface treatment.

[0183] Example 8. Evaluation of lactoferrin bioactivity by zone of inhibition A zone of inhibition test was performed to evaluate the biological activity of API-E2 lactoferrin: plates containing E. coli were incubated with 100 μg / mL, 10 μg / mL, 1 μg / mL, and 0.1 μg / mL of API-E2 lactoferrin added to paper circles and allowed to spread out.

[0184] As shown in Figure 14, complete inhibition was found in the diffusion zone at 100 μg / mL and 10 μg / mL API-E2 lactoferrin, with a limited inhibition zone at 1 μg / mL, demonstrating the antibacterial bioactivity of API-E2 lactoferrin. No inhibition zone was observed at 0.1 μg / mL.

[0185] Example 9. Evaluation of lactoferrin bioactivity based on LPO activity An enzymatic assay for lactoperoxidase (LPO) activity has been developed utilizing a molecule that turns blue in the presence of peroxidase activity.

[0186] As shown in Figure 15, this gives a strong positive response for the purified LPO standard whose presence was verified by mass spectrometry (Figure 15#3). For API-E2 lactoferrin purified as described herein, LPO was not detected by mass spectrometry or by enzyme assay (Figure 15#1). However, for the commercially available Sigma laboratory grade standard (L9507), the contaminant LPO could be confirmed by mass spectrometry, but no positive enzyme reaction was found to occur (Figure 15#2). This indicates that lactoferrin produced with the previous methodology resulted in a total loss of protein bioactivity when treated under these conditions. However, with the method described herein, whenever LPO could be detected by mass spectrometry, the corresponding enzyme activity was also observed. Only in samples where LPO was completely or nearly completely removed from the lactoferrin (mass spectrometry undetectable), no enzyme activity was observed. This indicates both that the API-E2 lactoferrin product achieves very high purity, and that the purification method described herein broadly retains protein activity throughout the purification process.

[0187] Equivalent While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

[0188] Incorporation by Reference All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A pharmaceutical composition comprising lactoferrin, the pharmaceutical composition being formulated for delivery to the oral cavity, nasal cavity, wounds, ocular cavity, and / or gastrointestinal tract.

2. The pharmaceutical composition is formulated for delivery to the oral or nasal cavity, and optionally (a) the pharmaceutical composition is formulated for retention of the lactoferrin in the buccal mucosa; (b) the pharmaceutical composition is formulated for delivery to the extracellular matrix (ECM) of a cell; (c) the pharmaceutical composition (i) tablets, optionally formulated as orally dissolving or disintegrating tablets; (ii) spray; (iii) gum; (iv) a thin film; or (v) lozenges and / or (d) the oral or nasal cavity includes the pharynx.

3. The pharmaceutical composition is formulated for delivery to a wound, and optionally (a) the pharmaceutical composition is formulated for delivery to skin tissue, ocular tissue, connective tissue, mucosal tissue, gastrointestinal tissue, and / or pulmonary tissue; (b) the wound comprises an open wound or ulcer, and optionally the ulcer is venous or arterial; (c) the wound is selected from the group consisting of a burn, a bite, a cut, an infection, an ulceration, and combinations thereof; and optionally (i) the burn is selected from the group consisting of thermal burns, chemical burns, electrical burns, radiation burns, friction burns, and combinations thereof; and / or (ii) the cut is selected from the group consisting of a puncture wound, a laceration, an abrasion, an incision, a cut, and combinations thereof; (d) the pharmaceutical composition comprises a bioadhesive; and / or (e) The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated in a delivery format selected from the group consisting of a gel foam, a hydrogel, an injection dressing, a powder, a spray, and an inhalation format.

4. The pharmaceutical composition is formulated for delivery to the gastrointestinal tract, and optionally (a) the pharmaceutical composition is formulated in a delivery format that includes microencapsulation; and / or (b) The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated in a delivery format that includes an enteric coating.

5. The pharmaceutical composition comprises: (a) excipients; (b) sodium steryl humate, carbomer, sodium hydroxide, glycerin, sodium benzoate, benzoic acid, citric acid, and / or combinations thereof; (c) xylitol, sorbitol, mannitol, maltitol, sugar alcohols, sucrolose, and / or combinations thereof; (d) isomalt, microcrystalline cellulose, sodium carboxymethylcellulose, and / or combinations thereof; (e) powdered sugar, gum base, corn syrup, gum arabic, and / or combinations thereof; (f) HiG PWD-04, encapsulated powdered flavor, anhydrous or natural caffeine, encapsulated sucralose or sucralose, encapsulated acesulfame-K and aspartame, aspartame, acesulfame-K, triacetin, silicon dioxide, and / or combinations thereof; (g) a coagulant, optionally comprising thrombin, amylopectin, kaolin, and / or combinations thereof; and / or (h) an astringent, optionally selected from the group consisting of alum, acacia, sage, yarrow, witch hazel, bayberry, distilled vinegar, persimmon, green tea, black tea, citric acid, cranberry juice and / or extract, grapefruit juice and / or extract, inorganic acids, and combinations thereof; 2. The pharmaceutical composition of claim 1, comprising: (a) the lactoferrin is bovine; and / or (b) The pharmaceutical composition of claim 1, wherein the lactoferrin has not been chemically, enzymatically, acidically, and / or heat-treated, and optionally the lactoferrin has not been heat-treated at a temperature of 50°C or higher, 51°C or higher, 52°C or higher, 53°C or higher, 54°C or higher, or 55°C or higher.

7. Purified lactoferrin (a) circular dichroism; and / or (b) Differential Scanning Calorimetry (DSC) 2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises a native conformation as assessed by HPLC, optionally wherein the native conformation comprises an apolactoferrin conformation and / or a hololactoferrin conformation, and optionally wherein the apolactoferrin conformation has a peak melting temperature of 60.2±0.8°C and / or the hololactoferrin conformation has a peak melting temperature of 88.38±0.8°C.

8. 2. The pharmaceutical composition of claim 1, wherein the purified lactoferrin is capable of binding iron, and optionally at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% of the purified lactoferrin is capable of binding iron, and / or the ability to bind iron is assessed by DSC.

9. The purified lactoferrin (a) comprising a post-translational modification, optionally wherein the post-translational modification comprises glycosylation; and / or 10. The pharmaceutical composition of claim 1, comprising (b) an average molecular weight of at least 79,000 to 86,000 Da.

10. The purified lactoferrin is (a) dried, optionally the purified lactoferrin is dried by freeze-drying / lyophilization, fluidized bed drying, or low-temperature spray drying; and / or (b) The pharmaceutical composition of claim 1, which remains in liquid form throughout the purification of lactoferrin.

11. The composition further comprises molecular iron, and optionally (a) the purified lactoferrin is complexed with an iron molecule, optionally comprising Fe 2+ or Fe 3+ ; and / or 2. The pharmaceutical composition of claim 1, wherein (b) the purified lactoferrin is complexed with copper, zinc, manganese, and / or gallium molecules.

12. 2. The pharmaceutical composition of claim 1, wherein the composition contains endotoxin at a level of 5 EU / kg or less.

13. The lactoferrin is purified from natural dairy products, and optionally (a) the natural dairy product has not been processed prior to the purification of the lactoferrin; and / or (b) the natural dairy product has not been chemically, enzymatically, acidically or thermally treated prior to the purification of the lactoferrin, and optionally the natural dairy product has not been heat treated at a temperature of 50°C or higher, 51°C or higher, 52°C or higher, 53°C or higher, 54°C or higher, or 55°C or higher prior to the purification of the lactoferrin.

14. (a) the lactoferrin is purified from a natural dairy product that has been separated into skim milk and cream prior to purification of the lactoferrin, and optionally the separation into skim milk and cream includes cold bowl separation; and / or (b) the lactoferrin is purified from a natural dairy product that has been acid-treated prior to purification of the lactoferrin, optionally the acid treatment including removal of insoluble casein, and optionally the acid treatment being at a pH of 4.0 or greater.

15. A method for producing the pharmaceutical composition of any one of claims 1 to 14, comprising: (a) one or more steps selected from the group consisting of chromatography, filtration, and drying, or each of the steps of chromatography, filtration, and drying; and / or (b) Pressing into a dosage form A method comprising:

16. 15. A pharmaceutical composition according to any one of claims 1 to 14 for use in a method for treating a disease or condition, said method comprising administering said pharmaceutical composition to a subject, and optionally (a) the disease or condition is selected from the group consisting of a pathogenic disease, a gastrointestinal (GI) infection, a wound infection, an allergic condition, an inflammatory condition, and combinations thereof, and optionally the disease or condition is a pathogenic disease and the subject has been diagnosed with an infection by a pathogen; (b) said administration is prophylactic, and optionally said subject (i) at risk of exposure to a pathogen; or (ii) has been exposed to a pathogen; and / or (c) the pathogen is (i) Microorganisms transmitted orally and / or nasally; (ii) airborne microorganisms; (iii) a virus, optionally comprising a coronavirus, optionally wherein the coronavirus is SARS-CoV-2; or (iv) bacteria or fungi A pharmaceutical composition comprising:

17. 15. A pharmaceutical composition according to any one of claims 1 to 14 for use in a method for reducing the risk of a pathogenic disease, said method comprising administering said pharmaceutical composition to a subject, and optionally (a) reducing the risk of pathogenic disease comprises administering the pharmaceutical composition to a wound of the subject; (b) the subject has been exposed to a pathogen and / or has been diagnosed with an infection by a pathogen; and / or (c) the pathogen is (i) Microorganisms transmitted orally and / or nasally; (ii) airborne microorganisms; (iii) a virus, optionally comprising a coronavirus, optionally wherein the coronavirus is SARS-CoV-2; or (iv) bacteria or fungi A pharmaceutical composition comprising:

18. 15. A pharmaceutical composition according to any one of claims 1 to 14 for use in a method for reducing the risk of infection, said method comprising administering said pharmaceutical composition to a subject, optionally said subject having a wound at risk of said infection.

19. 15. A pharmaceutical composition according to any one of claims 1 to 14 for use in a method for promoting wound healing in tissue, said method comprising administering said pharmaceutical composition to a wound in tissue of a subject, and optionally (a) the wound is selected from the group consisting of a burn, a cut, an infection, an ulceration, and combinations thereof; and optionally (i) the burn is selected from the group consisting of thermal burns, chemical burns, electrical burns, radiation burns, friction burns, and combinations thereof; or (ii) the cut is selected from the group consisting of a puncture wound, a laceration, an abrasion, an incision, a cut, and combinations thereof; (b) the tissue is skin tissue, lung tissue, or a combination thereof; and / or (c) the method comprises treating a disease or condition selected from the group consisting of a pathogenic disease, a gastrointestinal (GI) infection, a wound infection, an allergic condition, an inflammatory condition, and combinations thereof, and optionally treating the disease or condition comprises prophylactic treatment of the disease or condition.

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