Purified lactoferrin composition and method of use

Abstract

Compositions containing lactoferrin (in particular pharmaceutical formulations containing lactoferrin purified from raw natural milk sources) are provided. Methods for producing the same and its use are also provided. Pharmaceutical compositions containing lactoferrin are provided herein, and the pharmaceutical compositions are formulated for delivery to non-human subjects. In some embodiments, the non-human subjects are domesticated animals. In some embodiments, the domesticated animals are horses, cattle, pigs, sheep, goats, chickens, or camels. In some embodiments, the non-human subjects are pets. In some embodiments, the pets are cats or dogs.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority of U.S. Provisional Patent Application No. 63 / 486,437, filed on February 22, 2023, the entire disclosure of which is hereby incorporated by reference for all purposes.

[0002] Sequence Listing This application contains a sequence listing that was electronically submitted in XML format, which is hereby incorporated by reference in its entirety. The XML file generated on August 30, 2022, is named HYA - 001WO_SL.xml and has a size of 3,493 bytes. [[ID=!15]]

Background Art

[0003] Lactoferrin has been investigated in the context of its antimicrobial properties. However, preparations of lactoferrin typically are derived from pre - processed and / or pre - treated dairy products (such as pasteurized milk sources) or are produced recombinantly. Such production strategies can result in changes in the properties of lactoferrin (such as denaturation, reduction in bioactivity, reduction in iron - binding ability, alteration of sugar chain addition, and / or lack of retention of post - translational modifications, etc.) compared to those found in raw and / or untreated milk sources. Additionally, available purified lactoferrin products generally contain significant impurities.

[0004] Preparations and formulations of lactoferrin [generally including lactoferrin without impurities (such as other proteins, enzymes, endotoxins, prions, etc.)] directed towards retaining the native bioactivity of lactoferrin [especially pharmaceutically acceptable (such as FDA - registrable) preparations and formulations of lactoferrin for use in the fields of wound care and other applications] do not exist in the art.

Summary of the Invention

Means for Solving the Problems

[0005] It should be noted that in the above translation, the "!15" in the "Background Art" part is a possible error in the original text. It might be just a mis - typing or an incorrect tag usage. If it's a real error, it should be corrected according to the actual content requirements. Pharmaceutical compositions comprising lactoferrin are provided herein, and the pharmaceutical compositions are formulated for delivery to non-human subjects. In some embodiments, the non-human subjects are domesticated animals. In some embodiments, the domesticated animals are horses, cattle, pigs, sheep, goats, chickens, or camels. In some embodiments, the non-human subjects are pets. In some embodiments, the pets are cats or dogs.

[0006] In some embodiments, the pharmaceutical composition is formulated as animal feed, animal water, animal chew, pet food additive, or animal milk substitute.

[0007] In some embodiments, the pharmaceutical composition is formulated for topical administration. In some embodiments, the formulation for topical administration comprises a powder. In some embodiments, the powder comprises a lyophilized powder. In some embodiments, the formulation for topical administration comprises a teat dip.

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

[0009] In some embodiments, the pharmaceutical composition is formulated for injection into non-human animals.

[0010] In some embodiments, the pharmaceutical composition is formulated in an inhalation format. In some embodiments, the inhalation format is a nebulizer, inhaler, aerosol, or spray format.

[0011] In some embodiments, the pharmaceutical composition is formulated for delivery to a wound. In some embodiments, the pharmaceutical composition is formulated for delivery to skin tissue, eye tissue, connective tissue, mucous membrane tissue, gastrointestinal tissue and / or lung tissue. In some embodiments, the wound includes an open wound or an ulcer. In some embodiments, the ulcer is venous or arterial. In some embodiments, the wound is selected from the group consisting of burns, bites, cuts, infections, ulceration, and combinations thereof. In some embodiments, the infection is mastitis. In some embodiments, the burn is selected from the group consisting of thermal burns, chemical burns, electric burns, radiation burns, friction burns, and combinations thereof. In some embodiments, the cut is selected from the group consisting of punctures, lacerations, abrasions, incisions, severances, and combinations thereof. In some embodiments, the incision includes a surgical incision.

[0012] In some embodiments, the pharmaceutical composition includes a bioadhesive. In some embodiments, the pharmaceutical composition is formulated in a delivery format selected from the group consisting of gel foams, hydrogels, injected bandages, powders, sprays, and inhalation formats.

[0013] In some embodiments, the pharmaceutical composition is formulated for delivery to the gastrointestinal tract. In some embodiments, the pharmaceutical composition is formulated in a delivery format comprising lactoferrin, microencapsulation, microbeads, or an inert substance. In some embodiments, lactoferrin is produced via fluid bed drying to produce powder or microbeads via granulation, encapsulation, coating, or aggregation. In some embodiments, the pharmaceutical composition is formulated in a delivery format comprising an enteric coating. In some embodiments, the pharmaceutical composition is formulated for dental administration. In some embodiments, the formulation for dental administration is an oral spray, animal chew, or pet food additive.

[0014] Furthermore, pharmaceutical compositions containing lactoferrin are provided herein, and these pharmaceutical compositions are formulated for dental administration. In some embodiments, formulations for dental administration include toothpaste, mouthwash, floss, oral spray, effervescent tablets, mouth props, retainers, oral packing materials, or dental instrument disinfectants.

[0015] Furthermore, pharmaceutical compositions containing lactoferrin are provided herein, and these pharmaceutical compositions are formulated for delivery to the eye. In some embodiments, the formulation for delivery to the eye includes eye drops, contact lens solutions, eye washes, or eye sprays.

[0016] Furthermore, compositions comprising lactoferrin are provided herein, and the compositions are comprised of reproductive health formats. In some embodiments, the reproductive health format comprises a lubricant, a condom, a gel, an injected wipe, or an injected pad.

[0017] Furthermore, compositions comprising lactoferrin are provided herein, which are formulated for disinfection of surfaces. In some embodiments, surfaces include medical surfaces, hospital instruments, surgical instruments, surgical implants, dental instruments, food preparation surfaces, skin, nipples, or flesh surfaces.

[0018] Furthermore, compositions comprising lactoferrin are provided herein, which are formulated for use as food or in food products. In some embodiments, food and / or food products include animal feed, milk substitutes, colostrum substitutes, water, beverages, or pre-packaged foods.

[0019] Furthermore, compositions containing lactoferrin are provided herein, and these compositions are formulated as cosmetics. In some embodiments, the cosmetics include creams, lotions, sunscreens, or sprays.

[0020] In some embodiments, a pharmaceutical composition or composition includes an excipient.

[0021] In some embodiments, the pharmaceutical composition or composition comprises sodium stearyl fumarate, 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 alcohols, 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 flavoring substance, anhydrous caffeine or natural caffeine, encapsulated sucralose or sucralose, encapsulated acesulfame K and encapsulated aspartame, aspartame, acesulfame K, triacetin, silicon dioxide, and / or combinations thereof.

[0022] In some embodiments, the pharmaceutical composition or composition comprises a coagulating substance. In some embodiments, the coagulating substance comprises thrombin, amylopectin, kaolin, and / or a combination thereof.

[0023] In some embodiments, the pharmaceutical composition or composition comprises an astringent substance. In some embodiments, the astringent substance 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.

[0024] In some embodiments, lactoferrin is of bovine origin. In some embodiments, lactoferrin is untreated. In some embodiments, lactoferrin has not been chemically treated, enzymatically treated, acid treated, and / or heat treated. In some embodiments, lactoferrin has not been heat treated. In some embodiments, 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. In some embodiments, lactoferrin has not been heat treated at a temperature of 55°C or higher.

[0025] In some embodiments, purified lactoferrin comprises a native conformation assayed by circular dichroism.

[0026] In some embodiments, purified lactoferrin comprises a native conformation assayed by differential scanning calorimetry (DSC). In some embodiments, the native conformation comprises an apo-lactoferrin conformation and / or a holo-lactoferrin conformation. In some embodiments, the apo-lactoferrin conformation has a melting temperature peak of 60.2 ± 0.8°C and / or the holo-lactoferrin conformation has a melting temperature peak of 88.38 ± 0.8°C.

[0027] In some embodiments, purified lactoferrin is capable of binding iron. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the purified lactoferrin is capable of binding iron. In some embodiments, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the purified lactoferrin is capable of binding 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 the purified lactoferrin is capable of binding iron. In some embodiments, the ability to bind iron is assayed by DSC.

[0028] In some embodiments, the purified lactoferrin comprises post-translational modifications. In some embodiments, the post-translational modifications comprise glycosylation.

[0029] In some embodiments, the purified lactoferrin comprises an average molecular weight of at least 79,000 to 86,000 Da.

[0030] In some embodiments, the purified lactoferrin is dried. In some embodiments, the purified lactoferrin is dried by freeze-drying / lyophilization, fluid bed drying, or low-temperature spray drying. In some embodiments, the purified lactoferrin remains in liquid form via lactoferrin purification.

[0031] In some embodiments, the composition further comprises iron molecules. In some embodiments, the purified lactoferrin is complexed with iron molecules. In some embodiments, the iron molecules comprise Fe2+ or Fe3+. In some embodiments, the purified lactoferrin is complexed with copper molecules, zinc molecules, manganese molecules, and / or gallium molecules. In some embodiments, the purified lactoferrin is complexed with zinc molecules.

[0032] In some embodiments, the composition comprises endotoxin at a level of 5 EU / kg or less.

[0033] In some embodiments, lactoferrin is purified from untreated natural dairy products. In some embodiments, the natural dairy products are not processed before the purification of lactoferrin. In some embodiments, the natural dairy products are not chemically treated, enzymatically treated, acidically treated, or heat-treated before the purification of lactoferrin. In some embodiments, the natural dairy products are not heat-treated before the purification of lactoferrin. In some embodiments, the heat treatment includes temperatures 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 includes temperatures of 55°C or higher.

[0034] In some embodiments, lactoferrin is purified from natural dairy products separated into skim milk and cream prior to lactoferrin purification. In some embodiments, the separation into skim milk and cream includes cold bowl separation. In some embodiments, lactoferrin is purified from natural dairy products that have been acid-treated prior to lactoferrin purification. In some embodiments, the acid treatment includes the removal of insoluble casein. In some embodiments, the acid treatment may be at a pH of 4.0 or higher.

[0035] In some embodiments, lactoferrin is present in concentrations of 0.1–0.5 mg / ml, 0.125–0.5 mg / ml, 0.1–1 mg / ml, 0.1–0.25 mg / ml, or 0.125–0.25 mg / ml. In some embodiments, lactoferrin is present in concentrations of 0.125–0.5 mg / ml. In some embodiments, lactoferrin is present in concentrations of 1 mg / ml or less.

[0036] Methods for producing any of the pharmaceutical compositions described herein or any one of the compositions are also 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. In some embodiments, the method comprises pressing into a drug dosage form.

[0037] A method for treating a disease or condition, comprising administering any one of the pharmaceutical compositions or compositions described herein, is also provided herein. In some embodiments, the disease or condition is selected from the group consisting of pathogenic diseases, gastrointestinal (GI) infections, wound infections, allergic conditions, inflammatory conditions, and combinations thereof. In some embodiments, the disease or condition is mastitis.

[0038] A method for treating a pathogenic disease is also provided herein, comprising administering a pharmaceutical composition or any one of the compositions described herein to a subject. In some embodiments, the administration is prophylactic. In some embodiments, the subject is at risk of exposure to the pathogen. In some embodiments, the subject has been exposed to the pathogen. In some embodiments, the subject has been diagnosed with an infection by the pathogen.

[0039] Methods for reducing the risk of pathogenic disease, comprising administering any one of the pharmaceutical compositions or compositions described herein to a subject, are also provided herein. In some embodiments, the reduction of the risk of pathogenic disease comprises administering the pharmaceutical composition or the composition to a wound in a subject. In some embodiments, the administration of the pharmaceutical composition or the composition comprises topical administration. In some embodiments, topical administration comprises the administration of a powder. In some embodiments, the powder comprises lyophilized lactoferrin. In some embodiments, the formulation for topical administration comprises microbeads. In some embodiments, the microbeads comprises fluid-bed dried microbeads. In some embodiments, the fluid-bed dried microbeads are produced via granulation, encapsulation, coating, or aggregation. In some embodiments, the subject is exposed to a pathogen and / or diagnosed with an infection by the pathogen. In some embodiments, the pathogen comprises microorganisms transmitted orally and / or nasally. In some embodiments, the pathogen comprises airborne microorganisms. In some embodiments, the pathogen comprises viruses. In some embodiments, the virus comprises respiratory viruses. In some embodiments, the pathogen includes bacteria or fungi.

[0040] In some embodiments, the treatment of a disease or condition includes the prophylactic treatment of the disease or condition.

[0041] Methods for reducing the risk of infection are also provided herein, comprising administering a pharmaceutical composition or any one of the compositions described herein to a subject. In some embodiments, the subject has a wound that is at risk of infection. In some embodiments, the administration of the pharmaceutical composition or composition comprises topical administration. In some embodiments, topical administration comprises the administration of a powder, microbeads, or inert substance containing lactoferrin. In some embodiments, the powder comprises lyophilized lactoferrin. In some embodiments, the lactoferrin comprises fluid-bed dried lactoferrin. In some embodiments, the lactoferrin is produced via fluid-bed drying and generates a powder or microbeads via granulation, encapsulation, coating, or aggregation.

[0042] Methods for promoting wound healing of tissue, comprising administering the above pharmaceutical composition or any one of the compositions to a wound in the tissue of interest, are also provided herein. In some embodiments, the wound is selected from the group consisting of burns, cuts, infections, ulceration, and combinations thereof. In some embodiments, the burn is selected from the group consisting of thermal burns, chemical burns, electrical burns, radiation burns, friction burns, and combinations thereof. In some embodiments, the cut is selected from the group consisting of punctures, lacerations, abrasions, incisions, severances, and combinations thereof. In some embodiments, the tissue is skin, lung tissue, or combinations thereof. In some embodiments, the incision is a surgical incision. In some embodiments, the method comprises treating a disease or condition selected from the group consisting of pathogenic diseases, gastrointestinal (GI) infections, wound infections, allergic conditions, inflammatory conditions, and combinations thereof. In some embodiments, the treatment of the disease or condition comprises reducing pain and / or inflammation. In some embodiments, the treatment of the disease or condition comprises prophylactic treatment of the disease or condition. In some embodiments, the pharmaceutical composition or administration of the composition includes topical administration to a wound. In some embodiments, the topical administration includes administration of a powder. In some embodiments, the powder comprises lyophilized lactoferrin. In some embodiments, the lactoferrin comprises fluid-bed dried lactoferrin. In some embodiments, the lactoferrin is produced via fluid-bed drying and then granulated, encapsulated, coated, or aggregated to produce a powder or microbeads.

[0043] A method for disinfecting a surface, comprising applying a pharmaceutical composition or any one of the compositions described herein to the surface, is also provided herein. In some embodiments, the surface includes medical surfaces, hospital instruments, surgical instruments, surgical implants, dental instruments, food preparation surfaces, skin, nipples, or flesh surfaces. In some embodiments, skin includes wounds. In some embodiments, disinfection can inhibit microbial growth. In some embodiments, inhibition of microbial growth includes inhibiting or preventing biofilm formation.

[0044] In some embodiments, lactoferrin is present in concentrations of 0.1–0.5 mg / ml, 0.125–0.5 mg / ml, 0.1–1 mg / ml, 0.1–0.25 mg / ml, or 0.125–0.25 mg / ml. In some embodiments, lactoferrin is present in concentrations of 0.125–0.5 mg / ml. In some embodiments, lactoferrin is present in concentrations of 1 mg / ml or less. [Brief explanation of the drawing]

[0045] [Figure 1] Specific embodiments of the lactoferrin purification process described herein are illustrated. Briefly, raw milk (untreated milk, e.g., not chemically, enzymatically, acidically, or heat-treated before lactoferrin purification) is (1) diverted before entry into an industrial-standard milk processing workflow; (2) flowed through an ion exchange resin / column, with the eluate typically returned to a standard milk processing workflow (if requested / required by the raw milk producer), and the lactoferrin-containing eluate is collected; (3) the collected eluate is filtered; and (4) the purified lactoferrin is processed.

[0046] [Figure 2A] The MALDI-TOF (approximately 18kDa to 100kDa) purity assessment of lactoferrin produced according to Example 1 is shown. The raw material used was raw colostrum (RC).

[0047] [Figure 2B] The MALDI-TOF (approximately 18kDa to 100kDa) purity assessment of lactoferrin produced according to Example 1 is shown. The raw material used was raw whole milk (RM).

[0048] [Figure 3A] This shows the MALDI-TOF (approximately 18kDa to 100kDa) purity assessment of over-the-counter (OTC) lactoferrin supplements.

[0049] [Figure 3B] This shows the MALDI-TOF (approximately 18kDa to 100kDa) purity assessment of purchased laboratory-grade lactoferrin.

[0050] [Figure 3C] This shows the MALDI-TOF (approximately 18kDa to 100kDa) purity assessment of purchased laboratory-grade lactoferrin.

[0051] [Figure 4] The purity assessment of lactoferrin produced according to Example 1 is shown using Orbitrap Velos mass spectrometry.

[0052] [Figure 5A] The ELISA purity assessment of lactoferrin produced according to Example 1 and purchased laboratory reagent grade lactoferrin is shown.

[0053] [Figure 5B] This shows the lactoferrin purity assessed by HPLC.

[0054] [Figure 6] Immunofluorescence (IF) imaging shows lactoferrin enrichment in the extracellular matrix (ECM).

[0055] [Figure 7]This shows the quantification of lactoferrin enrichment in ECM.

[0056] [Figure 8] Immunofluorescence (IF) imaging of primary buccal mucosa cells demonstrates lactoferrin enrichment in the extracellular matrix (ECM).

[0057] [Figure 9] This shows a SarsCoV-2 CPE reduction assay in the presence of API-E2.

[0058] [Figure 10] The differential scanning calorimetry (DSC) assay plots assessing API-E2 lactoferrin (10 mg / mL), including the heat-treated version, are shown.

[0059] [Figure 11A] This shows DSC assay plots assessing API-E2 lactoferrin in the absence and presence of excess iron.

[0060] [Figure 11B] The DSC assay plots for assessing laboratory-grade standard lactoferrin and commercially available supplement-grade lactoferrin are superimposed on the API-E2 lactoferrin plot (see Figure 10).

[0061] [Figure 12] This paper demonstrates the assessment of lactoferrin's biological activity against salivary bacteria as an evaluation of pH over time in the presence of API-E2.

[0062] [Figure 13] The images show untreated chicken (left) or chicken treated with API-E2 (right) after being inoculated with human salivary bacteria and left at 37°C for 6 days.

[0063] [Figure 14]This indicates that an inhibition zone test was performed to assess the biological activity of API-E2 lactoferrin against E. coli.

[0064] [Figure 15] An outline of lactoperoxidase (LPO) activity and results for various lactoferrin samples, including API-E2, are shown.

[0065] [Figure 16] This shows relative bacterial growth in the presence of different lactoferrin sources.

[0066] [Figure 17] This shows relative bacterial growth over time in the presence of different lactoferrin sources.

[0067] [Figure 18A] This paper quantifies the inhibition of biofilm formation in the presence of different lactoferrin sources.

[0068] [Figure 18B] This shows a representative standard crystal violet assay plate for assessing the inhibition of biofilm formation in the presence of different lactoferrin sources.

[0069] [Figure 19] The survival curves of untreated mice and lactoferrin-treated (Lf) mice after wounding are shown.

[0070] [Figure 20] This shows wound healing assessment in pigs after treatment with different lactoferrin sources. Pigs were treated with Lactea-Lf (second from the left panel), bacitracin (second from the right panel), or silver sulfadiazine cream (far right panel). The far left panel shows exemplary wounds immediately after preparation.

[0071] [Figure 21]This paper presents a case study of cattle with large infectious chronic wounds before (left panel) and after (right panel) treatment with Lactea lactoferrin.

[0072] [Figure 22] This shows differential scanning calorimetry (DSC) assessments of different lactoferrin sources.

[0073] [Figure 23A] This document presents representative ITCs for evaluating the binding of Sigma Aldrich lactoferrin L9507 (research standard) and Lactea-Lf to heparan sulfate.

[0074] [Figure 23B] This shows a typical ITC for assessing integration with LPS, including competitive integration with HS. [Modes for carrying out the invention]

[0075] definition Unless otherwise specified, terms used in the claims and specification are defined as set forth below.

[0076] When used interchangeably herein, “unprocessed dairy products” or “unprocessed dairy products containing lactoferrin” refers to natural liquid lactation products collected directly from mammals, produced by the mammary glands of mammals, and to which no 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) have been applied.

[0077] When used interchangeably herein, “untreated dairy products” or “dairy products containing untreated lactoferrin” means dairy products that have not undergone any processing steps (e.g., chemical processing, enzymatic processing, acid processing, and / or heat processing) but have undergone one or more possible mechanical processing steps (e.g., filtration, column / resin purification, and / or milk separation).

[0078] Unless otherwise stated or made clear from the context, the term “about” as used herein is understood to mean within the normal tolerance range in the art (e.g., within two standard deviations of the mean). “About” may be understood to mean 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, in some cases, be deemed to be modified by the term “about,” in which case it will be clear from the context that the scope covered by the modification is consistent with the applicability of the invention and the limitations of the claims.

[0079] The 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), the homology alignment algorithm of Needleman & Wunsch, J.Mol.Biol.48:443 (1970), 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 (in the Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Dr., Madison, Wis.))), or by visual inspection (see Ausubel et al. in general).

[0080] 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 analysis is publicly available through the National Center for Biotechnology Information.

[0081] Lactoferrin Compositions comprising purified lactoferrin are provided herein. Generally, as used herein, lactoferrin refers to a purified form of mammalian lactoferrin obtained from unprocessed dairy products (e.g., raw milk).

[0082] The lactoferrin of the compositions purified herein is typically bovine, and is purified, for example, from bovine milk sources. Exemplary bovine lactoferrin (bLF) molecules include, but are not limited to, those described in CAS Registry No. 146897-68-9 and in 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).An exemplary, non-restrictive, full-length amino acid sequence of bovine (Bos taurus) lactoferrin is provided below by: (SEQ ID NO: 1; GenBank accession number AAA30610.1).

[0083] Lactoferrin may be a fragment of full-length lactoferrin (e.g., SEQ ID NO: 1). The fragment may include a biologically active fragment. As used herein, “biologically active” means a protein having one or more of the biological activities of the corresponding native protein, such biological activities include, but are not limited to, enzymatic activity, antimicrobial activity (e.g., antibacterial activity, antifungal activity, and / or antiviral activity), iron binding / sequencing activity, immunomodulatory behavior (e.g., anti-inflammatory activity), growth regulation, and cell surface affinity, wound healing, or any other activity of lactoferrin described herein or known in the art. For example, lactoferrin is typically secreted, and a biologically active fragment may be a secreted format (e.g., a “processed” fragment of lactoferrin lacking a signal peptide). As an exemplary example, lactoferrin represented by SEQ ID NO: 1 contains the signal peptide MKLFVPALLSLGALGLCLA (SEQ ID NO: 2; amino acids 1-19 of SEQ ID NO: 1). Therefore, a biologically active fragment of lactoferrin could be lactoferrin lacking the signal peptide (e.g., amino acids 20-708 of SEQ ID NO: 1).

[0084] Lactoferrin may be a lactoferrin isoform (such as lactoferrin alpha (LFα) isoform, lactoferrin beta (LFβ) isoform, or lactoferrin gamma (LFγ) isoform).

[0085] Lactoferrin may have at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as the amino acid sequence shown as SEQ ID NO: 1 or its biologically active fragment (e.g., a secreted form such as amino acids 20-708 of SEQ ID NO: 1 lacking the signal peptide). Lactoferrin may have at least 95% of the same amino acid sequence as the amino acid sequence shown as SEQ ID NO: 1 or its biologically active fragment. Lactoferrin may have at least 96% of the same amino acid sequence as the amino acid sequence shown as SEQ ID NO: 1 or its biologically active fragment. Lactoferrin may have at least 97% of the same amino acid sequence as the amino acid sequence shown as SEQ ID NO: 1 or its biologically active fragment. Lactoferrin may have an amino acid sequence that is at least 98% identical to the amino acid sequence shown as Sequence ID No. 1 or its biologically active fragment. Lactoferrin may have an amino acid sequence that is at least 99% identical to the amino acid sequence shown as Sequence ID No. 1 or its biologically active fragment. Lactoferrin may have an amino acid sequence that is at least 99.5% identical to the amino acid sequence shown as Sequence ID No. 1 or its biologically active fragment.

[0086] Lactoferrin may have conservative substitutions. “Conservative substitution” or “conservative amino acid substitution” refers to a substitution of an amino acid with a chemically or functionally similar amino acid. Conservative substitutions are well known in the art, as described, for example, in Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) WH Freeman & Co., New York, NY (incorporated herein by reference for all purposes).

[0087] Post-translation modification Lactoferrin found in unprocessed dairy products as a raw material typically contains post-translational modifications. While not 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 found in natural milk sources used for lactoferrin purification. For example, the purification strategies and methods described herein reduce, minimize, or eliminate chemical treatments, enzymatic treatments, acid treatments, heat treatments (e.g., pasteurization), and / or other treatments that may destroy native post-translational modifications.

[0088] Post-translational modifications may be modifications that contribute to the biological activity of lactoferrin. Generally, recombinant lactoferrin, typically produced in exogenous expression systems (such as bacteria or yeast), lacks post-translational modifications and / or post-translational modifications of natively produced lactoferrin. Post-translational modifications include, but are not limited to, glycosylation, phosphorylation, and acetylation. Post-translational modifications may include glycosylation (e.g., N-linked glycosylation), such as glycosylation at asparagine 233, 281, 368, 476, and / or 545, and such glycosylation may include acetylneuraminic acid, galactose, mannose, fucose, N-acetylglucosamine, and / or N-acetylgalactosamine. Post-translational modifications may be naturally occurring and / or non-natural (e.g., modifications after purification, such as by in vitro methods known to those skilled in the art). Post-translational modifications may include the processing of full-length lactoferrin described above (e.g., removal of signal sequences).

[0089] Unmodified secreted lactoferrin typically has a molecular weight of approximately 78 kDa. Native post-translational modifications can result in molecular weights ranging up to approximately 86 kDa. Purified lactoferrin may have a molecular weight greater than approximately 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. Purified lactoferrin may have a molecular weight of 82-85 kDa.

[0090] Methods for assessing post-translational modifications are known to those skilled 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).

[0091] Protein conformation The conformational state of lactoferrin found in unprocessed dairy products is typically considered to be the native conformation of lactoferrin. Various treatments of milk sources typically used for lactoferrin purification (such as pasteurization) are generally considered to be capable of denaturing the protein. While we do not wish to be bound by theory, the purification strategies and methods described herein are designed to reduce, minimize, or eliminate the destruction of the native conformation, with reference to the conformational states found in natural milk sources used for lactoferrin purification. For example, the purification strategies and methods described herein reduce, minimize, or eliminate chemical treatments, enzymatic treatments, acid treatments, heat treatments (e.g., pasteurization), and / or other treatments that may denature lactoferrin.

[0092] Methods for determining the conformation are known to those skilled in the art and include circular dichroism, X-ray crystallography, or antibody-mediated methods (e.g., the use of antibodies that recognize the conformation and / or non-denaturing Western blot analysis).

[0093] Iron complex formation Lactoferrin contains two iron-binding domains (also called globular lobes). While we do not wish to be bound by theory, iron-binding properties may mediate and / or influence antimicrobial biological activity (such as killing microorganisms, preventing microbial invasion, chelation, elimination of microorganisms, and / or inhibiting their growth).

[0094] Iron-bound lactoferrin is called hololactoferrin, and iron-free lactoferrin is called apolactoferrin. In some cases, they may differ in biological activity (such as their antimicrobial activity) or other properties (such as increased resistance to heat-induced changes in hololactoferrin compared to apolactoferrin). Lactoferrin in unprocessed dairy products is typically found within a defined ratio of iron-free to iron-bound form. For example, in bovine milk, lactoferrin generally contains 20-30% in the iron-bound form, and in human milk, generally 6-8% is in the iron-bound form. Purified lactoferrin in the compositions herein may contain a defined range of iron-bound hololactoferrin. Purified lactoferrin may contain 20-30% hololactoferrin. Purified lactoferrin may contain 6-8% hololactoferrin. Purified lactoferrin may contain more than 30% hololactoferrin. Purified lactoferrin may contain less than 6% hololactoferrin. Purified lactoferrin may contain at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% hololactoferrin. Purified lactoferrin may contain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% hololactoferrin. Purified lactoferrin may contain 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 be 100% hololactoferrin. Purified lactoferrin may contain less than 20% hololactoferrin. Purified lactoferrin may contain more than 8% hololactoferrin. Purified lactoferrin may contain less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% hololactoferrin. Purified lactoferrin may contain less than 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% hololactoferrin. Purified lactoferrin may be 100% iron-free apolactoferrin.

[0095] Differential scanning calorimetry (DSC) can be used to assess the ability of purified lactoferrin to become hololactoferrin, for example, to assess the ability of purified lactoferrin to bind iron. For example, the ability to bind iron can be assessed by adding an excess of iron in the presence of purified lactoferrin and then performing DSC to assess the relative peaks associated with apolactoferrin and hololactoferrin. Purified lactoferrin and / or drug formulations may contain at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the purified lactoferrin that is capable of binding iron. Purified lactoferrin and / or drug formulations may contain at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the purified lactoferrin that is capable of binding iron, as assessed by DSC. Purified lactoferrin and / or drug formulations may contain purified lactoferrin in which at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the purified lactoferrin is capable of iron binding. Purified lactoferrin and / or drug formulations may contain purified lactoferrin in which at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the purified lactoferrin is capable of iron binding, as assessed by DSC. Purified lactoferrin and / or drug formulations may contain purified lactoferrin in which 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 iron binding. Purified lactoferrin and / or drug formulations may contain purified lactoferrin in which 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 iron binding, as assessed by DSC.Purified lactoferrin and / or drug formulations may contain 100% iron-binding-capable purified lactoferrin, as assessed by DSC (e.g., the only observable peak in the presence of excess iron is the hololactoferrin-related peak).

[0096] Differential scanning calorimetry (DSC) may be used to assess the melting temperature peaks of purified lactoferrin (particularly those of apolactoferrin and / or hololactoferrin). Purified lactoferrin and / or drug formulations may contain purified lactoferrin with a melting temperature peak associated with apolactoferrin of 60.2 ± 0.8 °C. Purified lactoferrin and / or drug formulations may contain purified lactoferrin with a melting temperature peak associated with hololactoferrin of 88.38 ± 0.8 °C.

[0097] In the hololactoferrin form, lactoferrin typically contains two ferric iron (Fe) compounds found in natural milk sources. 3+ Purified lactoferrin in the compositions herein may bind to metal ions other than ferric ions (including, but not limited to, copper, zinc, manganese, and / or gallium). Purified lactoferrin may bind to zinc ions. Purified lactoferrin may bind to Fe 2+ It can bind to ions. Purified lactoferrin bound to metal ions other than ferric ions may be any hololactoferrin form or apolactoferrin form of ferric-bound lactoferrin described herein, for example, in any defined ratio of hololactoferrin form to apolactoferrin form as described herein.

[0098] The conformations of lactoferrin (e.g., hololactoferrin conformation and apolactoferrin conformation) vary in level with metal ions (e.g., Fe 3+ They can be saturated with iron. For example, apolactoferrin is typically saturated with less than 5% iron ions, while hololactoferrin is typically saturated with about 100% iron. Bovine lactoferrin in unprocessed dairy products is typically 15-20% iron saturated.

[0099] 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 (addition or removal, etc.) the concentrations of ferric ions, other metal ions, and / or non-ferric-based molecules and / or iron-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)] (incorporated herein by reference for all purposes)).

[0100] Methods for assessing the ability of purified lactoferrin to bind metal ions are known to those skilled in the art, and include chemical analysis and / or absorption spectroscopy. For example, although we do not wish to be bound by theory, processing processes (e.g., those typically used in industrial purification) and / or recombinant production processes can alter the metal-binding ability of lactoferrin (e.g., through denaturation of the iron-binding domain).

[0101] Methods for determining the ratio of hololactoferrin form to apolactoferrin form are known to those skilled in the art and include chemical analysis and / or absorption spectroscopy (as described, for example, in Majka et al. [Analytical and Bioanalytical Chemistry volume 405, pages 5191-5200 (2013)] (incorporated herein by reference for all purposes)).

[0102] In non-limiting examples, differential scanning calorimetry (DSC) provides both a method for assessing the ability of purified lactoferrin to bind metal ions and a method for assessing the ratio of hololactoferrin form to apolactoferrin form. The DSC method is known to those skilled in the art.

[0103] Refining of dairy products The purification method can reduce, minimize, or eliminate chemical treatment, enzymatic treatment, acid treatment, and / or heat treatment. The purification method can reduce chemical treatment, enzymatic treatment, acid treatment, and / or heat treatment. The purification method can minimize chemical treatment, enzymatic treatment, acid treatment, and / or heat treatment. The purification method can eliminate chemical treatment, enzymatic treatment, acid treatment, and / or heat treatment. The purification method can reduce, minimize, or eliminate chemical treatment. The purification method can reduce, minimize, or eliminate enzymatic treatment. The purification method can reduce, minimize, or eliminate acid treatment. The purification method can reduce, minimize, or eliminate heat treatment. The purification method can reduce each of the chemical treatment, enzymatic treatment, acid treatment, and heat treatment. The purification method can minimize each of the chemical treatment, enzymatic treatment, acid treatment, and heat treatment. The purification method can eliminate each of the chemical treatment, enzymatic treatment, acid treatment, and heat treatment. The purification method can eliminate the effects of chemical treatment, enzymatic treatment, and acid treatment. The purification method can eliminate the effects of chemical treatment, enzymatic treatment, and heat treatment.

[0104] In general, the methods provided herein for the production of purified lactoferrin do not involve heat treatments typical of industrial purification processes (e.g., pasteurization) that can destroy (e.g., denature) the native conformation, with reference to the conformations found in the natural milk source used for lactoferrin purification. Typical heat treatments used in industrial purification processes may be about 63°C or higher. Heat treatments may be 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 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 method may include carrying out the process at a temperature that reduces, minimizes, or eliminates the destruction of the native conformation compared to typical heat treatments in industrial purification processes, with reference to the conformations found in the natural milk source used for lactoferrin purification. The purification method may include carrying out the process at a temperature that reduces, minimizes, or eliminates the reduction in lactoferrin bioactivity compared to typical heat treatments in industrial purification processes, with reference to the bioactivity found in the natural milk source used for lactoferrin purification. The purification method may include receiving the natural milk source at a refrigerated temperature (e.g., below 15°C, such as between 2 and 15°C). The purification methods described herein may include heat treatment below 50°C. For example, the milk source may be warmed between one or more purification steps (e.g., to a temperature above 37°C but not above 55°C). The 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. Heating may include temperatures above 37°C and below 55°C. Heating may include temperatures above 40°C and below 55°C. Heating may include temperatures above 45°C and below 55°C. Heating may include temperatures above 50°C and below 55°C. Heating may include temperatures above 37°C and temperatures below 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C.Heating may include temperatures above 40°C, and temperatures below 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C. Heating may include temperatures above 45°C, and temperatures below 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C. The purification methods described herein may include heat treatment at temperatures 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 at temperatures below 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C. Maintaining the temperature during purification may include maintaining the temperature throughout the purification process. Maintaining the temperature during purification may include maintaining the temperature at one or more individual steps of the purification process (e.g., chromatography, filtration, and / or drying steps). The maintained temperature may include fluctuating temperatures (e.g., different temperature ranges) specific to one or more individual steps of the purification process.

[0105] Purification methods may include acid treatment. While we do not wish to be bound by theory, acid treatment may be used for the removal of casein (e.g., treating a natural milk source or derivative at a pH that can render casein insoluble in solution before lactoferrin purification). In general, the acid treatments provided herein for the production of purified lactoferrin do not include acid treatments that disrupt the native conformation (e.g., denaturing lactoferrin) and / or reduce the biological activity of lactoferrin, with reference to the conformational states or biological activity found in the natural milk sources used for lactoferrin purification, respectively. Purification methods may include acid treatment at a pH of 4.0 or higher. Purification methods may include acid treatment at a pH of 3.0 or higher.

[0106] The purification method may include chromatography. The purification method may include ion exchange chromatography. Methods of chromatography (such as ion exchange chromatography) are known to those skilled in the art. The purification methods described herein will generally include cation exchange chromatography. In addition to cation exchange chromatography, the purification method may include one or more additional purification processes, such as ion exchange chromatography (including both cation exchange chromatography and anion exchange chromatography in any order and / or separated). Resins and matrices for ion exchange chromatography are known in the art. For example, cation exchange resins include, but are not limited to, polymethacrylate matrices and agarose matrices. The purification method may include high-pressure liquid chromatography (HPLC).

[0107] Chromatographic methods generally include one or more equilibration and / or regeneration steps. Exemplary, non-limiting examples of equilibration and regeneration steps include (1) rinsing with reverse osmosis water; (2) rinsing with chemically pure 1 M NaCl; and (3) re-rinsing with reverse osmosis water.

[0108] Chromatographic methods generally involve a filling step. Generally, the volume filled is based on the predetermined binding capacity of the resin and an estimate of the native lactoferrin content of the raw milk feed material.

[0109] Chromatographic methods generally involve one or more elution steps in ion-exchange chromatography (e.g., elution of purified lactoferrin from resin / column). Elution methods are known to those skilled in the art. Elution methods may include two or more elution steps. While we do not wish to be bound by theory, multiple elution steps can be used to first elute contaminants (e.g., other products besides lactoferrin) and then the desired product (e.g., lactoferrin).

[0110] The elution method may include two or more elution steps at different salt concentrations. While we do not wish to be bound by theory, one or more initial elution steps (e.g., elution steps prior to the elution step containing the desired purified lactoferrin) may be performed to remove unwanted proteins and other contaminants.

[0111] The elution method may include a first elution step of a 0.2–0.7 M chemically pure NaCl solution. Generally, the first elution step of a 0.2–0.7 M chemically pure NaCl solution is performed to remove unwanted proteins and other contaminants. While not wishing to be bound by theory, the first elution may be monitored for completion by ultraviolet-visible light spectroscopy and / or colorimetric assays to monitor for the presence of contaminants (such as lactoperoxidase) and other enzymes native to the raw milk feedstock. For example, the first elution step of a 0.2–0.7 M chemically pure NaCl solution may be performed until the absence of protein elution from the resin is detected by ultraviolet-visible light spectroscopy.

[0112] The elution method may include a first elution step of a 0.2 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.25 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.30 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.35 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.40 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.45 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.50 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.55 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.6 M 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 also include a first elution step of a chemically pure NaCl solution of less than 1 M.

[0113] The elution method may include a second elution step of a 1M chemically pure NaCl solution. Generally, elution using a 1M chemically pure NaCl solution will elute lactoferrin from the resin. The elution method may include a second elution step of approximately 1M chemically pure NaCl solution.

[0114] The elution method may include a first elution step of a 0.2-0.7 M chemically pure NaCl solution and a second elution step of a 1 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.2 M chemically pure NaCl solution and a second elution step of a 1 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.25 M chemically pure NaCl solution and a second elution step of a 1 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.3 M chemically pure NaCl solution and a second elution step of a 1 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.35 M chemically pure NaCl solution and a second elution step of a 1 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.40 M chemically pure NaCl solution and a second elution step of a 1 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.45 M chemically pure NaCl solution and a second elution step of a 1 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.50 M chemically pure NaCl solution and a second elution step of a 1 M chemically pure NaCl solution. The elution method may include a first elution step of a 0.55 M chemically pure NaCl solution and a second elution step of a 1 M chemically pure NaCl solution. The elution method may include a first elution step of a chemically pure NaCl solution of less than 1 M and a second elution step of a 1 M chemically pure NaCl solution.

[0115] The elution method may include a first elution step of a 0.25-0.7 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 0.20 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 0.25 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 0.30 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 0.35 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 0.40 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 0.45 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 0.50 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 0.55 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 0.60 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 0.65 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.

[0116] The elution method may include two or more elution steps at different pH levels. Elution gradient. 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 a salt elution gradient and a pH elution gradient.

[0117] For each of the above ion exchange chromatography steps (e.g., equilibration, regeneration and packing, and / or elution), a person skilled in the art will recognize that the appropriate liquid rate depends on the selection of, for example, the resin, apparatus, raw milk feed material, etc.

[0118] The purification method may include filtration. Filtration methods are known to those skilled in the art. The purification method may include microfiltration (typically referring to filtration using membrane pore sizes of 0.1 to 10 μm). Microfiltration may include membrane pore sizes of 1 to 10 μm. Microfiltration may include a membrane pore size of 10 μm. Microfiltration may include membrane pore sizes of 1 to 10 μm. Microfiltration may include membrane pore sizes of 0.1 to 1 μm. Microfiltration may include a membrane pore size of 0.1 μm. Microfiltration may include a membrane pore size of 1 μm. Microfiltration may include membrane pore sizes of 0.1, 0.2, 0.3, 0.3, 0.5, 0.6, 0.7, 0.8, 0.9, and / or 1 μm. Microfiltration may include ceramic filters.

[0119] The purification method may include ultrafiltration (typically referring to filtration using a membrane pore size of 0.01–0.1 μm). An ultrafiltration system may also be referred to by a molecular weight cutoff size designed for separation based on the size between the permeate and the retaining solution. Ultrafiltration systems may include systems of 5–30 kDa. Ultrafiltration systems may include systems of 5 kDa. Ultrafiltration systems may include systems of 10 kDa. Ultrafiltration systems may include systems of 15 kDa. Ultrafiltration systems may include systems of 20 kDa. Ultrafiltration systems may include systems of 25 kDa. Ultra-ultrafiltration systems may include systems of 30 kDa.

[0120] The purification method may include both microfiltration and ultrafiltration, comprising one or more additional purification processes in any order and / or separated. The purification method may include multiple microfiltration steps and / or ultrafiltration steps, comprising one or more additional purification processes in any order and / or separated. As an exemplary non-limiting example, a first pre-filtration microfiltration step (e.g., with a 10 μm filter) may be used before ion exchange chromatography, a second microfiltration step (e.g., with a 0.1–1.4 μm filter) may be used following ion exchange chromatography, and then an ultrafiltration step (e.g., with 5–30 kDa) may be used.

[0121] A purification method may include a combination of chromatography and filtration, comprising one or more additional purification processes, separated in any order. A purification method may include a combination of multiple chromatography steps and / or filtration steps, comprising one or more additional purification processes, separated in any order. A purification method may include a combination of microfiltration, ultrafiltration, and ion exchange chromatography, comprising one or more additional purification processes, separated in any order. In an exemplary non-limiting example, a purification method may include ion exchange chromatography (including elution), followed by microfiltration, and then ultrafiltration. In another exemplary non-limiting example, a purification method may include microfiltration, followed by ion exchange chromatography (including elution), followed by additional microfiltration, and then ultrafiltration.

[0122] Purification methods may include separating raw dairy products into dairy derivatives (such as separating a natural milk source into skim milk and cream). For example, raw dairy products may be separated into dairy derivatives before chromatography and / or filtration. Methods for separating raw dairy products into dairy derivatives are known to those skilled in the art and include, but are not limited to, cold bowl separation.

[0123] Following the purification of lactoferrin, the purified product may be dried. Generally, the drying methods provided do not involve processes that disrupt the native conformation (e.g., denaturing lactoferrin) and / or reduce the biological activity of lactoferrin, with reference to the conformational states or bioactivity found in the natural milk source used for lactoferrin purification. Methods for drying lactoferrin are known to those skilled in the art and include, but are not limited to, freeze-drying / lyophilization, fluidized bed drying (e.g., as fluidized bed dried microbeads), fluidization (e.g., fluidization onto an inert material), and / or low-temperature spray drying (e.g., spray drying onto an inert material). In non-limiting exemplary examples, purified lactoferrin is freeze-dried / lyophilized into a powder.

[0124] Purity assessment Natural milk sources (such as bovine milk) typically contain several protein components in addition to lactoferrin (including, but not limited to, lactoperoxidase, lysozyme, casein, immunoglobulins, lactalbumin, and lactoglobulin). Natural milk sources may also contain other components (such as fats and endotoxins). Generally, the purification methods provided herein reduce, minimize, or eliminate components other than lactoferrin. While we do not wish to be bound by theory, the removal of one or more of the additional components may improve the biological activity and / or safety of lactoferrin.

[0125] This specification provides a lactoferrin composition having an increased percentage of lactoferrin by mass compared to the proportion present in dairy products containing unprocessed lactoferrin.

[0126] The percentage of lactoferrin can be assessed by mass spectrometry, including, but not limited to, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry and / or linear-trap quadrupole orbit-trap Velos mass spectrometry. Generally, assessment by mass spectrometry involves quantifying the area under the peak corresponding to lactoferrin and the area under the peak not corresponding to lactoferrin. In some examples, lactoferrin may be associated with multiple peaks (such as the ionization peak corresponding to lactoferrin). The peak corresponding to lactoferrin may include the peak corresponding to full-length post-translational modified (e.g., glycosylated) lactoferrin. The peak corresponding to full-length post-translational modified lactoferrin is generally in the range of approximately 80,000–86,000 m / z. The exact peak for post-translational modified full-length lactoferrin may vary, reflecting different glycosylation states, etc. In some examples, to be comprehensive, a peak with m / z between 79,000 and 90,000 may be determined to correspond to lactoferrin. The area under the peak not corresponding to lactoferrin includes all other peaks, with the possible exception of the ionization peak associated with lactoferrin around 41,500 m / z (e.g., with m / z between 41,000 and 42,000). The area under the peak not corresponding to lactoferrin may include peaks with m / z between 18,000 and 80,000 (other than those with m / z between 41,000 and 42,000). Specific comparisons can also be made between the peaks corresponding to lactoferrin and lactoperoxidase (e.g., peaks with m / z between 77,000 and 78,000). The area under the peak not corresponding to lactoferrin may include peaks with m / z between 18,000 and 45,000 (other than those with m / z between 41,000 and 42,000). Linear-trap quadrupole orbit-trap Velos mass spectrometry can also quantify the percentage of lactoferrin compared to other components in the sample. For example, quadrupole orbital Velos mass spectrometry can quantify the percentage of lactoferrin by determining the percentage of peptide spectral matches (PSMs).

[0127] The lactoferrin percentage can be assessed by liquid chromatography (e.g., high-performance liquid chromatography (HPLC)). Assessment by liquid chromatography may include quantifying the area under the peak corresponding to lactoferrin and the area under the peak not corresponding to lactoferrin.

[0128] Regarding assessments involving peak quantification, one or more sub-peak areas not corresponding to lactoferrin present in unprocessed lactoferrin-containing dairy products may be below the detection limit for purified lactoferrin compositions. In some instances, the sub-peak area not corresponding to each lactoferrin may be below the detection limit.

[0129] The percentage of lactoferrin can be assessed by enzyme-linked immunosorbent assay (ELISA). In some cases, ELISA can distinguish the percentage of lactoferrin in the native protein conformation (e.g., by using an antibody that specifically binds to the native protein conformation of lactoferrin).

[0130] Lactoperoxidase is a specific component typically present in unprocessed milk sources that should be reduced, minimized, or eliminated during the purification of lactoferrin. In addition, lactoperoxidase is frequently present in detectable amounts in purified lactoferrin compositions produced by typical industrial methods.

[0131] Lactoferrin compositions are provided herein, wherein at least 70% of the composition is purified lactoferrin. 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. Compositions include those in which at least 75% of the composition is purified lactoferrin. Compositions include those in which at least 80% of the composition is purified lactoferrin. Compositions include those in which at least 85% of the composition is purified lactoferrin. Compositions include those in which at least 90% 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 about 100% of the composition is purified lactoferrin. 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. The composition includes those in which at least 91% of the composition is purified lactoferrin. The composition includes those in which at least 92% of the composition is purified lactoferrin. The composition includes those in which at least 93% of the composition is purified lactoferrin. The composition includes those in which at least 94% of the composition is purified lactoferrin. The composition includes those in which at least 95% of the composition is purified lactoferrin. The composition includes those in which at least 96% of the composition is purified lactoferrin. The composition includes those in which at least 97% of the composition is purified lactoferrin. The composition includes those in which at least 98% of the composition is purified lactoferrin. The composition includes those in which at least 99% of the composition is purified lactoferrin.

[0132] This specification provides lactoferrin compositions having an increased lactoferrin:lactoperoxidase ratio compared to the ratio in dairy products containing unprocessed lactoferrin. The lactoferrin:lactoperoxidase ratio can be assessed according to methods known to those skilled in the art, such as purity assessment methods described herein (e.g., mass spectrometry, HPLC, and / or ELISA). For example, the assessment may include quantifying the area under the peak(s) corresponding to lactoferrin and the area under the peak(s) corresponding to lactoperoxidase.

[0133] Endotoxins present or potentially present in natural milk sources can be reduced, minimized, or eliminated. While we do not wish to be bound by theory, the removal of endotoxins may improve the safety of purified lactoferrin compositions. Methods for assessing endotoxin levels are known to those skilled in the art.

[0134] A method for assessing the purity of a purified lactoferrin composition (such as using any of the purity assessment methods described herein) is also provided herein.

[0135] Pharmaceutical compositions and other formats Pharmaceutical compositions comprising any one of the purified lactoferrin compositions described herein and one or more pharmaceutically acceptable excipients are provided herein.

[0136] As used herein, “pharmaceutical composition” encompasses a range of compositions suitable for administration to a subject (mammal, particularly humans, etc.). Generally, “pharmaceutical compositions” are sterile and preferably free from contaminants that could induce an undesirable response in the subject (e.g., the compounds in the pharmaceutical composition are pharmaceutical grade). Pharmaceutical compositions may be designed for administration to a subject or patient requiring administration via a number of different routes of administration, including, but not limited to, topical routes such as formulations for the skin (e.g., wounds), mucous membranes, respiratory tract, oral cavity (including gastrointestinal tract), and nasal cavity.

[0137] "Pharmacologically acceptable excipients," "pharmaceutically acceptable diluents," "pharmaceutically acceptable carriers," and "pharmaceutically acceptable adjuvants" mean excipients, diluents, carriers, and adjuvants that are generally safe, non-toxic, and not undesirable in either biological or otherwise, and that are useful in the preparation of pharmaceutical compositions, and include acceptable excipients, diluents, carriers, and adjuvants for veterinary and human pharmaceutical use. When used herein and in the claims, "pharmaceutically acceptable excipients, diluents, carriers, and adjuvants" includes one and more of such excipients, diluents, carriers, and adjuvants.

[0138] For pharmaceutical applications, lactoferrin may be incorporated into orally soluble formulations. Such formulations may include, but are not limited to, orally soluble tablets, lozenges, gums, oral thin films, or direct application of lactoferrin in excipient powders. Pharmaceutically acceptable excipients include, but are not limited to, sodium stearyl fumarate, citrate, xylitol (Xylisorb XTAB 240), sorbitol (Neosorb XTAB 200S or Neosorb P60 W), mannitol, maltitol (Sweetpearl P300 DC), and / or other sugar alcohols, as well as any combination thereof. While not wishing to be bound by theory, orally soluble formulations may enhance the retention of lactoferrin in the mouth and nasopharynx for localized targeting of lactoferrin. Orally soluble formulations may be prepared using techniques known in the art (e.g., fluidized bed drying or other drying techniques coupled to pressing the product into the final dosage form). Oral formulations may contain bioadhesives. Applicable bioadhesives are known to those skilled in the art and include those described in Duan et al. (Applications of Bioadhesives: A Mini Review; Front Bioeng Biotechnol. 2021; 9: 716035.) (incorporated herein by reference for all purposes). Oral formulations may contain astringents. Applicable astringents are known to those skilled in the art and 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 extracts, grapefruit juice and / or extracts, or inorganic acids.

[0139] Oral formulations may have specific applicability for gastrointestinal applications (including non-human (e.g., animal) applications). Oral formulations (including those for gastrointestinal applications) may include microencapsulation of lactoferrin. Oral formulations (including those for gastrointestinal applications) may include microencapsulation of pharmaceutical compositions containing any one of the purified lactoferrin compositions described herein (including enteric-coated formulations). Oral formulations (including those for gastrointestinal applications) may include microencapsulation of pharmaceutical compositions containing any one of the purified lactoferrin compositions combined with one or more additional activators (e.g., enteric-coated multi-component formulations). Microencapsulation formats are known to those skilled in the art, such as those described in Singh et al. (Microencapsulation: A promising technique for controlled drug delivery; Res Pharm Sci. 2010 Jul-Dec; 5(2):65-77.) (incorporated herein by reference for all purposes).

[0140] For pharmaceutical applications aimed at wound healing (including wounds in non-human (e.g., animals)), lactoferrin may be incorporated into formulations that enhance contact between lactoferrin and the wound. A wound can be any damage to the tissue in question. Damage includes, but is not limited to, burns (e.g., thermal burns, chemical burns, electrical burns, radiation burns [ultraviolet or radiation, etc.], friction burns, etc.), cuts (e.g., punctures, lacerations, abrasions, incisions [surgical incisions, etc.], amputations, etc.), infections, and ulceration. A wound can include an open wound. A wound can include an ulcer (e.g., a venous ulcer or an arterial ulcer). Multiple forms of damage may coexist. For example, as an exemplary non-limiting example, damage may include both a burn with infection or a cut with infection. Tissues include, but are not limited to, skin and lung tissue. Damage to multiple tissues may coexist. For example, in exemplary and non-limiting examples, damage may encompass damage to both skin and lung tissue. Formulations that enhance contact between lactoferrin and wound may include bioadhesives. Applicable bioadhesives are known to those skilled in the art and are described herein, among others. Delivery formats for wound healing formulations include, but are not limited to, gel forms, hydrogels, injected bandages, powders, sprays, and inhalation formats (e.g., nebulizers, inhalers, and spray formats).

[0141] For pharmaceutical applications aimed at enhancing antimicrobial use, lactoferrin may be incorporated into formulations that enhance its antimicrobial activity. Antimicrobial use may include the treatment of active infections. Antimicrobial use may include the prophylactic prevention and / or treatment of infections. For example, the prophylactic prevention and / or treatment of infections may include application to wounds before infection results occur.

[0142] Formulations and / or delivery formats may enhance multiple uses and / or properties of lactoferrin. Non-limiting exemplary examples include formulations or delivery formats that enhance both the wound healing and antimicrobial properties of lactoferrin, and enable both increased cell proliferation and prevention of microbial biofilm formation.

[0143] Lactoferrin may be incorporated into formulations referred to as “medical foods,” typically considered foods for medicinal purposes. These applications include incorporation into gum or other chewable products for the slow release of lactoferrin. Medical food formulations (such as gum) may contain excipients (such as xylitol, powdered sugar, gum base, sorbitol, mannitol, sucralose, and / or corn syrup, and any combination thereof). For gum, lactoferrin may be incorporated either throughout the gum or as a coating on the outside of the gum. Medical food formulations may be prepared using techniques known in the art, such as fluidized bed drying or other drying techniques and final preparation with a gum base to form the final dosage form. Lactoferrin compositions may include formulations for use in other food formulations or food products (such as animal feed, milk substitutes, colostrum substitutes, water, beverages, or pre-packaged foods (e.g., ready-to-eat powders “MREs”)).

[0144] The formulation may contain sodium stearyl fumarate, carbomer, sodium hydroxide, glycerin, sodium benzoate, benzoic acid, citric acid, and / or combinations thereof. The formulation may contain xylitol, sorbitol, mannitol, maltitol, sugar alcohols, sucralose, and / or combinations thereof. The formulation may contain isomalt, microcrystalline cellulose, sodium carboxymethylcellulose, and / or combinations thereof. The formulation may contain powdered sugar, gum base, corn syrup, gum arabic, and / or combinations thereof. The formulation may contain HiG PWD-04, encapsulated powdered flavoring substances, anhydrous caffeine or natural caffeine, encapsulated sucralose or sucralose, encapsulated acesulfame K and encapsulated aspartame, aspartame, acesulfame K, triacetin, silicon dioxide, and / or combinations thereof.

[0145] The formulation may contain a coagulation substance, which may include, but is not limited to, thrombin, amylopectin, kaolin, and / or combinations thereof.

[0146] Pharmaceutical compositions (including orally soluble formulations and medical foods) may have a pH optimized to maximize the stability and activity of the protein. The dosage format (including the use of selected excipients) may contribute to enhancing the bioavailability, stability, and / or bioactivity of the protein.

[0147] Any of the pharmaceutical compositions described herein may take the format described herein, but compositions that are not essentially pharmaceuticals, such as cosmetics (e.g., cosmetic products for the skin or mouth), are also provided herein, and such cosmetics include, but are not limited to, creams, lotions, sunscreens, and sprays.

[0148] Pharmaceutical compositions may include compositions formulated for delivery to non-human subjects (e.g., for veterinary use). Non-human subjects may include, but are not limited to, domesticated animals such as horses, cattle, pigs, sheep, goats, chickens, or camels. Non-human subjects may include animals typically considered pets (e.g., cats or dogs). Exemplary, non-limiting examples of formats for delivery to non-human subjects include animal feed, animal chews, pet food additives (e.g., toppings for pet food), animal water, or animal milk substitutes (e.g., formulations for foals, calves, piglets, lambs, chicks, kittens, or puppies).

[0149] Formulations for delivery to non-human subjects may include formulations for topical administration (e.g., Teat Dip, particularly Teat Dip for the prophylaxis and / or treatment of mastitis). Formulations for delivery to non-human subjects may include powders (any of which are freeze-dried / lyophilized or fluid-bed dried powders) or microbeads produced via granulation, encapsulation, coating, or aggregation as described herein. Formulations for delivery to non-human subjects may include microbeads (fluid-bed dried microbeads, etc.). Formulations for delivery to non-human subjects may include any of the other pharmaceutical formats described herein (any of which are nasal sprays, injectable formats, and wound-directed formats as described herein).

[0150] Pharmaceutical compositions may include compositions formulated for dental administration (such as toothpaste, mouthwash, floss, oral spray, effervescent tablets, mouth props, retainers, oral packing materials, or dental instrument disinfectants). Pharmaceutical compositions may also include compositions formulated for dental administration in animal applications (such as formulations as oral sprays, animal chews, or pet food additives (e.g., pet food toppings)).

[0151] Pharmaceutical compositions may include compositions formulated for delivery to the eye (such as eye drops, contact lens solutions, eye washes, and eye sprays).

[0152] The composition may include those made up in a reproductive health format (such as lubricants, condoms, gels, injected wipes, injected pads, etc.).

[0153] The composition may include formulations for disinfecting surfaces (medical surfaces, hospital instruments, surgical instruments, surgical implants, dental instruments, food preparation surfaces, skin, nipples, and meat surfaces (e.g., chicken, pork, or beef)).

[0154] The composition may include substances capable of inhibiting microbial growth (including, but not limited to, inhibiting or preventing biofilm formation) on surfaces (e.g., any of the surfaces described herein). Inhibition of microbial growth can be assessed as a measure of inhibition and / or prevention of growth in liquid culture assays. Inhibition of microbial growth can be assessed as a measure of inhibition and / or prevention of biofilm formation in crystal violet assays, etc.

[0155] The composition (including, but not limited to, formulations capable of inhibiting or preventing biofilm formation) may include purified lactoferrin at concentrations of 0.1-0.5 mg / ml, 0.125-0.5 mg / ml, 0.1-1 mg / ml, 0.1-0.25 mg / ml, or 0.125-0.25 mg / ml. The composition may include purified lactoferrin at a concentration of 0.1-0.5 mg / ml. The composition may include purified lactoferrin at a concentration of 0.125-0.5 mg / ml. The composition may include purified lactoferrin at a concentration of 0.1-1 mg / ml. The composition may include purified lactoferrin at a concentration of 0.1-0.25 mg / ml. The composition may include purified lactoferrin at a concentration of 0.125-0.25 mg / ml. The composition may include purified lactoferrin at a concentration of 1 mg / ml or less.

[0156] The composition (including, but not limited to, formulations capable of inhibiting or preventing biofilm formation) may include purified lactoferrin at concentrations of approximately 0.1–0.5 mg / ml, approximately 0.125–0.5 mg / ml, approximately 0.1–1 mg / ml, approximately 0.1–0.25 mg / ml, or approximately 0.125–0.25 mg / ml. The composition may include purified lactoferrin at a concentration of approximately 0.1–0.5 mg / ml. The composition may include purified lactoferrin at a concentration of approximately 0.125–0.5 mg / ml. The composition may include purified lactoferrin at a concentration of approximately 0.1–1 mg / ml. The composition may include purified lactoferrin at a concentration of approximately 0.1–0.25 mg / ml. The composition may include purified lactoferrin at a concentration of approximately 0.125–0.25 mg / ml. The composition may include purified lactoferrin at a concentration of approximately 1 mg / ml or less.

[0157] Treatment and risk prevention / reduction methods A method for treating a disease or condition is provided herein by administering a therapeutically effective amount of any of the pharmaceutical compositions described herein (such as any one of the pharmaceutical compositions formulated for delivery to the oral cavity, nasal cavity, or other tissues (e.g., skin, or lung tissue)). Diseases or conditions include, but are not limited to, pathogenic diseases, gastrointestinal (GI) infections, wound infections, allergies, and inflammatory conditions.

[0158] Methods for modulating an immune response (e.g., enhancing anti-inflammatory activity) by administering any one of the purified lactoferrin compositions described herein in a therapeutically effective amount (including administering any of the pharmaceutical compositions described herein) are also provided herein. The term “modulate” encompasses maintaining biological activity, inhibiting (partially or completely) biological activity, and stimulating / activating (partially or completely) biological activity. The term also encompasses decreasing or increasing (e.g., promoting) biological activity. For example, administration of a therapeutically effective amount of purified lactoferrin may reduce inflammation (e.g., in the context of inflammatory diseases) by enhancing anti-inflammatory activity. In another example, administration of a therapeutically effective amount of purified lactoferrin may enhance analgesic properties (e.g., reducing pain and / or inflammation).

[0159] As used herein, the terms “treatment,” “to treat,” and similar terms refer to obtaining a desired pharmacological and / or physiological effect (such as prevention, risk reduction, relief and / or elimination of an infection, such as a viral infection, a fungal (e.g., yeast) infection, or a bacterial infection). Treatment can be prophylactic in that it completely or partially prevents the disease or its symptoms, and / or therapeutic in that it partially or completely cures the disease and / or any side effects caused by the disease. Treatment covers any treatment of disease in mammals (especially in humans) and includes (a) treating prophylactically (completely or partially) the occurrence of the disease (or symptoms of the disease) in subjects who may be predisposed to the disease but have not yet been diagnosed with the disease (e.g., subjects at risk of infection); (b) inhibiting the disease (e.g., eliminating the infection and / or reducing the infection to below the undetectable level); and (c) mitigating the disease (e.g., reducing the microbial burden associated with the infection).

[0160] Methods for reducing the risk of pathogenic disease, comprising administering any of the pharmaceutical compositions or compositions described herein to a subject, are also provided herein. For example, reducing the risk of pathogenic disease may include, but is not limited to, administering the pharmaceutical compositions or compositions described herein to a wound at risk for pathogenic disease (e.g., infection by a pathogen). A subject at risk for pathogenic disease may be exposed to a pathogen. A subject at risk for pathogenic disease may have been diagnosed with an infection by a pathogen. Risk reduction may include administering the pharmaceutical compositions or compositions described herein that reduce the risk of pathogenic disease compared to a subject that has not received the pharmaceutical composition or composition.

[0161] Exemplary, non-limiting examples include wounds (in animals, etc.) which may be treated with the pharmaceutical compositions or compositions described herein, including treating active pathogenic diseases and / or reducing the risk of pathogenic diseases, such as by topical administration of purified lactoferrin described herein as a lyophilized powder. Other forms of lactoferrin may also be used, such as fluidized bed dried lactoferrin powder, or microbeads produced via granulation, encapsulation, coating, or aggregation (e.g., fluidized bed dried microbeads).

[0162] A method for treating a subject identified as being at risk of a pathogenic disease, comprising administering to the subject a pharmaceutical composition or any composition described herein, thereby reducing the risk of the pathogenic disease, is also provided herein.

[0163] The "therapeutic dose" or "effective dose" means the amount of a compound sufficient to achieve such treatment for a disease, condition, or disorder when administered to a mammal or other subject for the treatment of that disease, condition, or disorder. The "therapeutic dose" will vary depending on the compound, the disease and its severity, as well as the age, weight, etc., of the subject being treated.

[0164] The target compound may be administered to the subject alone or in combination with additional activators. The terms “agent,” “compound,” and “drug” are used interchangeably herein. The method may further encompass the co-administration of a second agent (e.g., small molecules, antibodies, antibody fragments, antibody-drug conjugates, aptamers, proteins, antibiotics, antivirals, antimicrobial agents, antibacterial agents, antifungal agents, and / or vaccines) simultaneously or sequentially. In some embodiments, the method further encompasses the performance of radiotherapy on the subject.

[0165] The terms “co-administration” and “in combination” encompass the administration of two or more therapeutic agents simultaneously, in parallel, or consecutively without specific time constraints. In one embodiment, the agents are present in cells or the body of the subject for the same amount of time, or exert their biological or therapeutic effects simultaneously. In one embodiment, the therapeutic agents are in the same composition or unit dosing form. In other embodiments, the therapeutic agents are in separate compositions or unit dosing forms. When used herein, the term “unit dosing form” refers to a physically discontinuous unit suitable as a single dose for human and animal subjects, each unit containing a calculated predetermined amount of a compound (e.g., lactoferrin as described herein) in an amount sufficient to produce the desired effect in combination with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for unit dosing forms depend on the specific compound used and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host. In certain cases, a combination provides an enhanced effect compared to any of the components alone, and in some cases, a combination provides a hyperadditive or synergistic effect compared to the combined or additive effect of the components. For multiple medications, two drugs may be directly alternated, or, for example, two or more doses of one drug may be alternated with a single dose of another drug.

[0166] Methods for treating pathogenic diseases are also provided herein. The methods may include administering a pharmaceutical composition to a subject, such as 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 may be prophylactic. The subject may be at risk of exposure to a pathogen. For example, the risk of exposure to a pathogen may include, but is not limited to, administration to a wound where there is a risk of infection by the pathogen. The subject may have been exposed to a pathogen. The subject may have been diagnosed with an infection by a pathogen.

[0167] Diseases or conditions that may be considered include mastitis (especially infection-induced mastitis). Treatment of mastitis may include treating active mastitis or the prophylactic treatment of mastitis. Formulations for the treatment of mastitis in humans may include formulations for topical administration to the nipple. Formulations for the treatment of mastitis in veterinary applications (e.g., in livestock) may include formulations for topical administration (e.g., teat dip).

[0168] 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).

[0169] Methods for promoting wound healing (e.g., through enhanced anti-inflammatory activity, enhanced cell proliferation, and / or prevention of infection) are also provided herein by administering any one of the purified lactoferrin compositions described herein in a therapeutically effective amount (including administering any one of the pharmaceutical compositions described herein).

[0170] A method for disinfecting a surface by applying any one of the purified lactoferrin compositions described herein is also provided herein. Surfaces that can be sterilized include medical surfaces, hospital instruments, surgical instruments, dental instruments, food preparation surfaces, skin, nipples, or meat surfaces (e.g., chicken, pork, or beef).

[0171] Manufacturing method A method for any one of the purified lactoferrin compositions described herein (including any of the pharmaceutical compositions described herein) is provided herein. The method includes any one of the purification steps described in the section “Purification of Dairy Products,” and includes combinations of the steps described therein. Exemplary, non-limiting examples are described in the Examples section herein. [Examples]

[0172] The following are examples of specific embodiments for carrying out the present invention. These examples are presented for illustrative purposes only and are not intended to limit the scope of the present invention in any way. While efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantities, temperatures, etc.), a certain degree of experimental error and deviation should naturally be permitted.

[0173] The implementation of this invention will, unless otherwise indicated, utilize conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, within the scope of the skills of those skilled in the art. Such techniques are adequately described in the literature. See, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); ALLehninger, 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); and Carey and Sundberg, Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B (1992).

[0174] Example 1. Purification of lactoferrin As shown in Figure 1, lactoferrin was purified. Briefly, raw milk (untreated milk, e.g., not chemically, enzymatically, acidically, or heat-treated before lactoferrin purification) was (1) diverted before entry into an industrial-standard milk processing workflow; (2) flowed through an ion exchange resin / column, with the eluate typically returned to a standard milk processing workflow to collect the lactoferrin-containing eluate; (3) the collected eluate was filtered; and (4) the purified lactoferrin was processed. Notably, all other known lactoferrin purification processes typically begin with a milk source obtained after an industrial-standard milk processing workflow, which typically includes heat pasteurization. In addition, because a natural, unprocessed dairy product (e.g., raw milk) was used as the raw material, the lactoferrin contained reasonable post-translational modifications that potentially contribute to its biological activity, in contrast to recombinant lactoferrin.

[0175] Bovine lactoferrin was isolated and purified using a chromatography column to retain native post-translational modifications, glycosylation, and linked iron. The column was packed with a selected cation exchange resin (either a polymethacrylate matrix or agarose matrix) and equilibrated and regenerated by (1) rinsing with reverse osmosis water; (2) rinsing with chemically pure 1 M NaCl; and (3) re-rinsing with reverse osmosis water.

[0176] Raw, untreated, unprocessed whole cow's milk (less than 24 hours after milking) was obtained directly from dairy transport containers or silos before separation, defatting, heating, and / or pasteurization. Lactoferrin was purified from both raw colostrum (RC) and raw whole milk (RM).

[0177] The milk was filtered to remove larger particles using a 10 μm filter, heated above 37°C and maintained at a temperature below 63°C (generally considered the pasteurization start temperature), and then packed into a chromatography column. The volume packed was based on the predetermined binding capacity of the resin and an estimate of the native lactoferrin content of the raw milk feed material. During the packing process, all flow-through was returned to the pasteurizer's balance tank to return the plant supply to the point before separation and pasteurization.

[0178] Once filling was complete, the resin was rinsed with reverse osmosis water to remove any remaining milk compounds not bound to the resin. The first elution involved washing the resin with a chemically pure NaCl solution (0.2-0.7 M) and was completed by UV-Vis spectroscopic and colorimetric assays to monitor for the presence of contaminants (such as lactoperoxidase) and other enzymes native to the raw milk feedstock (i.e., the absence of protein elution from the resin). In particular, the first NaCl elution was carried out until lactoperoxidase, which is generally the major contaminant, was no longer present in the eluted fraction, as assessed by a peroxidase colorimetric assay using a peroxidase substrate for colorimetric analysis. The fraction from the first elution was held in an isolation container. The resin was then rinsed with reverse osmosis water. A second elution was carried out with 1 M NaCl to isolate lactoferrin. The second elution fraction was retained in an isolation container and then (1) filtered through a ceramic microfiltration filter (0.1–1.4 μm) and (2) concentrated using an ultrafiltration system (5–30 kDa).

[0179] Next, the desalted eluate is freeze-dried, or it may be directly passed into a fluidized bed dryer and dried on pharmaceutical-grade excipients. The purified lactoferrin produced according to the methods described herein is referred to as Hyacinth lactoferrin ("Hyacinth") or Lactea lactoferrin ("Lactea-Lf"); lactoferrin (only without the accompanying words "laboratory grade" or "commercially available supplement grade"); ODT-SC210; and API-E2. Various names may refer to purified lactoferrin produced according to variations of the methods described herein.

[0180] Example 2. Assessment of lactoferrin purity. The purified lactoferrin produced according to Example 1 was assessed for purity.

[0181] Typically, SDS-PAGE gels are used to assess the purity of reference-grade protein-derived products (including reference-grade lactoferrin). However, SDS-PAGE gels have a poor reputation for yielding exaggerated purity results depending on experimental conditions (Kurien and Scofield Methods Mol Biol. 2012;869:633-640). Therefore, purity was assessed using more sensitive methods such as mass spectrometry, HPLC, and ELISA.

[0182] Lactoferrin purity was assessed by MALDI-TOF mass spectrometry. The dried sample was dissolved in water at a concentration of 10 mg / mL. The dissolved sample was mixed with an equal volume of saturated sinapic acid in 50% acetonitrile containing 0.1% trifluoroacetic acid. The sample / matrix mixture (2 μL) was placed on an M1P 384 polished steel MALDI plate. MALDI mass spectra were acquired in cation 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 within these mass ranges using Protein Calibration Standard II (Bruker). MS spectra were analyzed using F1exAnalysis 3.4 (Bruker Daltonics, Billerica, MA).

[0183] The purity results assessed by MALDI-TOF (approximately 18 kDa to 100 kDa) for lactoferrin purified 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 dominant peak above approximately 80,000 m / z corresponds to glycosylated lactoferrin, while the peak at approximately 41,500 m / z corresponds to the ionization peak of lactoferrin. Notably, the typical contamination peak associated with lactoperoxidase (approximately 78,000 m / z) was below the detection limit. Quantification of the mass spectrometry profile revealed that over 75% of the determined relative area under the curve (AUC) for the peaks quantified in the 18 kDa–100 kDa range corresponded to the desired glycosylated lactoferrin peak at 80–85 kDa, and nearly 100% corresponded to lactoferrin when the ionization peak at approximately 41,500 m / z was included (the peaks selected for quantification, in other words, the true peaks judged to exceed the background noise, were determined by F1exAnalysis 3.4). Therefore, the results demonstrate that the purification process described herein produced highly pure lactoferrin from various raw natural dairy products. [Table 1A] [Table 1B]

[0184] The purity of the over-the-counter (OTC) lactoferrin supplement [Jarrow Formulas] (Figure 3A) and the lactoferrin source advertised as a laboratory reagent grade product [Sigma Bovine Colostrum Lactoferrin] (Figures 3B and 3C) was also assessed as described above by MALDI-TOF (approximately 18 kDa to 100 kDa) and quantified in Tables 2A, 2B, and 2C, respectively. In contrast to the lactoferrin produced according to Example 1, the mass spectrometry profiles revealed contaminating peaks (particularly lactoperoxidase) for both the OTC supplement and both laboratory reagent grade lactoferrin sources (see the peak in Figure 3B at 77806 m / z). Quantification of the mass spectrometry profiles revealed that only 25.3%, 52.3%, and 11.9% of the determined relative AUC for the peaks quantified in the 18 kDa to 100 kDa range corresponded to the desired glycosylated lactoferrin peak at 80–85 kDa. Even considering the lactoferrin ionization peak at approximately 41,500 m / z, only the combined relative AUCs of 33.5%, 66.4%, and 15.9%, respectively, corresponded to lactoferrin. Therefore, the results demonstrate that the purification process described herein produces lactoferrin of higher purity than available OTC supplements and laboratory reagent-grade lactoferrin sources. [Table 2A] [Table 2B] [Table 2C]

[0185] Lactoferrin purity was also assessed by linear trap quadrupole Orbitrap Velos mass spectrometry. As shown in Figure 4 and quantified in Table 3A, the Orbitrap Velos mass spectrometry profile for lactoferrin produced according to Example 1 revealed that over 80% of the identified peptide spectral matches (PSMs) corresponded to the desired lactoferrin. In contrast, as shown in Table 3B, the Orbitrap Velos mass spectrometry profile for the OTC supplement 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, the results demonstrate that the purification process described herein produces highly pure lactoferrin, which is of higher purity than other available lactoferrin sources. [Table 3A] [Table 3B]

[0186] Lactoferrin purity was also assessed by ELISA. As shown in Figure 5A, when the same amount of protein was packed by weight, the lactoferrin produced according to Example 1 ("Hyacinth") demonstrated a 30% increase in antibody binding compared to existing research standards. Therefore, the data demonstrate that the purified Hyacinth Proteins (API-E2) achieved higher purity and / or retained a greater fraction of the native conformational protein state compared to other research-grade reference products.

[0187] The purity of lactoferrin was assessed using an NaCl gradient by HPLC with a Thermo Fisher U3000 protein purification system along with a Tricorn 5 / 150 column packed with Cytiva BigBeads. As shown in Figure 5B, API-E2 processed by HPLC exhibits a single peak corresponding to lactoferrin, indicating approximately 100% purity.

[0188] The data demonstrate that the purification process of Example 1 achieved higher purity lactoferrin compared to existing available reagents, particularly in its ability to reduce contamination by lactoperoxidase.

[0189] Example 3. Assessment of lactoferrin activity by extracellular matrix association. The development of an infectious disease requires a multi-step process of viral progression. First, once the virus enters the host by binding to host heparan sulfate (HS) proteoglycans in the extracellular matrix (ECM), the virus targets cells, facilitating the binding of viral particles to specific receptors on the cell surface. Subsequently, the virus migrates internally and replicates within the host cell. While antiviral drugs typically focus on inhibiting either key viral replication proteins or specific receptors on the cell surface, the non-specific cell targeting mechanism of HS in ECM binding can also inhibit, interfere with, or prevent the virus from binding to target cells. Because this pathway is relatively non-specific and requires coating the ECM of exposed cells, conventional therapeutic agents utilizing this approach typically must be administered at relatively high local concentrations for maximum efficacy and are generally plagued by impurities and / or toxicity. Therefore, lactoferrin formulations of higher purity (e.g., pharmaceutical grade standards) would offer significantly reduced dosages. In addition, preparations that retain native functions (e.g., native conformation, post-translational modifications, iron-binding ability, etc.) will retain the efficacy of lactoferrin compared 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 with greater capacity than other lactoferrin products.

[0190] To assess the biological activity of lactoferrin (particularly its ability as an antiviral agent), its binding to the extracellular matrix (ECM) was evaluated. Purified lactoferrin was produced according to Example 1. Caco-2 cells (human-derived cell cultures) were plated onto coverslips and grown for 3 days, resulting in cell division and proliferation, and the ECM was fully developed. Purified lactoferrin was then added to the cells at varying concentrations at 37°C for 2 hours, allowing it to bind as theoretically required. Immunofluorescence (IF) imaging was used to directly observe the localization of the API, staining both the API and E-cadherin (a cell membrane marker).

[0191] As shown in Figure 6, enrichment of the ECM with purified lactoferrin was observed with increasing concentration. Primary localization within the cell was also observed at low concentrations, but slight localization of lactoferrin into the intracellular space was expected, given that lactoferrin possesses receptors that enable its known intracellular roles and uptake. To quantify API localization, both the absolute intensity of the fluorescence signal immediately outside / along the E-cadherin mark and the absolute intensity of the fluorescence signal inside the cell were measured. As shown in Figure 7, quantification of lactoferrin localization demonstrated enrichment in the ECM with increasing concentration. The results indicate that purified lactoferrin exhibited biological activity to bind to the ECM.

[0192] Next, primary buccal mucosa cells were cultured according to a standard protocol (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 commercially available laboratory-grade standards, lactoferrin was added to the medium at a final concentration of 100 μg / mL at 37°C for 1 hour. This is the approximate concentration at which API-E2 lactoferrin has been previously found to begin saturating the binding of cultured cells. Immunofluorescence imaging was performed as previously described. As shown in Figure 8, API-E2 lactoferrin showed strong binding along the ECM of buccal mucosa cells under these conditions (top row). However, commercially available laboratory-grade standards showed only limited ECM association of lactoferrin under the same conditions (bottom row). The results strongly suggest that API-E2 lactoferrin possesses more potent and important bioactivity in relation to host cell ECM association (which is necessary for both the protein's antiviral activity and its antibiotic activity in reducing biofilms).

[0193] Example 4. SARS-CoV-2 antiviral activity. The antiviral activity of purified lactoferrin against SARS-CoV-2 was assessed by performing a cytopathic effect (CPE) reduction assay.

[0194] Vero E6 cells were inoculated into 96-well cell culture plates at a cell density of 80–100%. Cells were incubated at 37°C for 2 hours using 3-fold serial dilutions of SC210, starting at a concentration of 1 mg / mL. Subsequently, cells were either mock-infected (for analysis of the cytotoxicity of the compound) or infected at a MOI of 0.001 in 150 μl of culture medium containing purified lactoferrin, reagent-grade lactoferrin, or remdesivir produced according to Example 1. Three days after infection, cell viability was assessed by staining cells with neutral red dye for 2 hours, extracting the dye in 50:50 Sorensen citrate buffer / ethanol for 30 minutes, and determining the EC50 (50% effective antiviral concentration) by reading the optical density at OD540 nm. As shown in Figure 9 and quantified in Table 4, purified lactoferrin inhibited viral replication at lower concentrations than comparable research-grade reference products. The results demonstrate that purified lactoferrin produced according to Example 1 inhibited SARS-CoV-2 virus infection more effectively than reagent-grade lactoferrin, suggesting enhanced biological activity and / or purity. [Table 4]

[0195] Example 5. Assessment of lactoferrin biological activity by differential scanning calorimetry. Lactoferrin possesses several important biological activities related to its antimicrobial activity, including iron chelation and host cell binding. Natively, lactoferrin exists in both iron-bound (hololactoferrin) and iron-free (apolactoferrin) forms. Given lactoferrin's strong binding to iron, and without wanting to be constrained by theory, hololactoferrin must be substantially more stable than apolactoferrin.

[0196] Hololactoferrin and apolactoferrin morphology are assessed by monitoring the unfolding temperature of lactoferrin samples using a nanoDSC (TA Instruments, Lindon, UT) in accordance with a standard DSC protocol known to those skilled in the art (see, for example, Hinz et al. “MEASUREMENT AND ANALYSIS OF RESULTSOBTAINED ON BIOLOGICAL SUBSTANCES WITH DIFFERENTIAL SCANNING CALORIMETRY”; incorporated herein by reference for all purposes).

[0197] API-E2 lactoferrin (10 mg / mL) produced as described herein was assessed by DSC. As shown in Figure 10 (solid line), API-E2 lactoferrin exhibited two peaks from left to right: a larger peak corresponding to apolactoferrin and a smaller peak corresponding to hololactoferrin. The absence of other peaks indicates the absence of contaminants and impurities, and API-E2 can be assessed as approximately 100%. The results indicate that the presence of iron stabilized 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. In addition, the absence of other peaks also indicated the purity of API-E2 lactoferrin. The peak of API-E2 apolactoferrin had a Tm of 60.2 ± 0.8 °C and a ΔH of 466.1 ± 9.2 kJ / mol. The peak of API-E2 hololactoferrin has a Tm of 88.38 ± 0.8 °C and a ΔH of 632.7 ± 7.2 kJ / mol. Hololactoferrin natively accounts for an average of 8.74 ± 1.39% of total API-E2 lactoferrin, while apolactoferrin comprised the remainder in the tested samples. However, the percentage of hololactoferrin can naturally vary. API-E2 was calculated to contain substantially 100% pure lactoferrin using this technique. [Table 5-1]

[0198] The assay was also performed on heat-treated API-E2 lactoferrin. API-E2 lactoferrin was heated over a range of 50–100°C. Notably, the heat range used in the DSC assay is comparable to the temperatures typically used industrially before lactoferrin purification (e.g., above 63°C). As shown in Figure 10 (dashed line), heat-treated API-E2 lactoferrin demonstrated loss of both detectable peaks, indicating that both the apolactoferrin and hololactoferrin forms were completely denatured after heat treatment.

[0199] Further DSC assays were used to assess the biological activity of lactoferrin (particularly the iron-binding capacity of API-E2 lactoferrin). Iron chloride (FeCl3) was added to API-E2 lactoferrin in a 1000-fold molar excess. In the presence of excess iron, API-E2 lactoferrin would bind iron to its open iron-binding site. If API-E2 retains its biological activity throughout production, all apolactoferrin is expected to shift to a more stable hololactoferrin form, as determined by the presence of their corresponding DSC peaks. As shown in Figure 11A, API-E2 lactoferrin exhibited two peaks in the absence of excess iron: a larger peak on the left corresponding to apolactoferrin and a smaller peak on the right corresponding to hololactoferrin. Upon addition of excess iron, 100% of the available apolactoferrin shifted to the hololactoferrin form, as demonstrated by the single lactoferrin peak observed here (right-hand peak), without a detectable apolactoferrin peak. The results demonstrate that iron-binding biological activity is completely retained in API-E2 lactoferrin produced using the method described herein.

[0200] Laboratory-grade reference lactoferrin and commercially available supplement-grade lactoferrin were also assessed by DSC under the same conditions used to assess API-E2 above. As shown in Figure 11B, the peak for laboratory-grade reference lactoferrin was significantly smaller and less distinct than that observed for API-E2, and contained one extra peak attributable to impurities. The curve had a nonspecific upward slope, as is typically observed in products where highly denatured proteins are present in the sample. The estimated ratio of apolactoferrin peaks to hololactoferrin peaks was almost equal in this result, indicating a loss of apolactoferrin during processing. Similarly, both apolactoferrin and hololactoferrin peaks were denatured at lower temperatures (55°C and 85°C, respectively), demonstrating a higher degree of instability and loss of iron-binding capacity due to previous chemical, enzymatic, and / or heat treatments. As also shown in Figure 11B, commercially available supplement-grade lactoferrin (lower line) did not show a detectable peak, indicating that the product was either completely denatured or lacked the presence of lactoferrin molecules.

[0201] Example 6. Assessment of lactoferrin biological activity against salivary bacteria by pH test. Salivary bacteria (including Lactobacillus, Lactococcus, and Streptococcus mutans) produce acid during growth. This acidification is strongly associated with tooth decay, especially when it results in a pH below 5.5 (which is a clinically valid threshold). Slurries of these bacteria were treated with sucrose to stimulate growth and varying concentrations of API-E2 lactoferrin. Upon addition of sucrose and API-E2 lactoferrin, the pH was adjusted to 7 via titration, and the pH change was subsequently monitored over the next 6 hours.

[0202] As shown in Figure 12, in the presence of sucrose without treatment with API-E2 lactoferrin, salivary bacteria produced acid, resulting in a pH drop to below 5.5 within 4 hours. However, in the presence of sucrose with either 1 mg / mL or 10 mg / mL of API-E2 lactoferrin, the pH was maintained above 5.5 throughout the entire 6-hour test period. The results indicate that the presence of API-E2 reduced bacterial acid production in a concentration-dependent manner, demonstrating that API-E2 lactoferrin possesses antimicrobial activity.

[0203] Example 7. Assessment of lactoferrin bioactivity by surface treatment of chicken meat. Chicken breast was inoculated with human salivary bacteria (including Lactobacillus, Lactococcus, Streptococcus mutans, and Porphyromonas gingivalis) in the slurry described above in a pH test, and left at 37°C for 6 days.

[0204] As shown in Figure 13, without treatment, chicken breast was covered with bacterial growth at the end of the 6-day window. However, with treatment with 3 mg / mL of API-E2 lactoferrin, no detectable bacterial growth was observed after 6 days, demonstrating the antimicrobial activity of API-E2 lactoferrin after surface treatment.

[0205] Example 8. Assessment of lactoferrin biological activity using inhibition zones. The biological activity of API-E2 lactoferrin was assessed by performing inhibition zone tests. Plates containing E. coli were cultured with 100 μg / mL, 10 μg / mL, 1 μg / mL, and 0.1 μg / mL of API-E2 lactoferrin added to paper circles, allowing for diffusion to the outside.

[0206] As shown in Figure 14, API-E2 lactoferrin at concentrations of 100 μg / mL and 10 μg / mL completely inhibited the diffusion zone, and a limited inhibition zone was observed at 1 μg / mL, demonstrating the antimicrobial activity of API-E2 lactoferrin. No inhibition zone was observed at 0.1 μg / mL.

[0207] Example 9. Assessment of lactoferrin biological activity by LPO activity. Enzyme assays for lactoperoxidase (LPO) activity were developed using molecules that turn blue in the presence of peroxidase activity.

[0208] As shown in Figure 15, purified LPO references whose presence was verified by mass spectrometry yielded a strong positive response (Figure 15 #3). For API-E2 Lf purified as described herein, LPO was not detected by either mass spectrometry or enzyme assay (Figure 15 #1). However, for commercially available Sigma laboratory-grade references (L9507), while the contaminant LPO could be observed by mass spectrometry, it was found not to result in a positive enzymatic reaction (Figure 15 #2). This indicates that lactoferrin produced by prior methodologies resulted in an overall loss of the biological activity of proteins treated under these conditions. In the processes described herein, however, whenever LPO could be detected by mass spectrometry, the corresponding enzymatic activity was also observed. Enzymatic activity was not observed only in samples where LPO was completely or nearly completely removed from lactoferrin (not detectable by mass spectrometry). This indicates both that the API-E2 lactoferrin product achieves very high purity and that the purification process described herein broadly preserves protein activity throughout the purification steps.

[0209] Example 10. Assessment of lactoferrin biological activity by inhibiting bacterial growth. As an analytical baseline for assessing lactoferrin (Lf) activity, 1 mg / mL of Lf was added to 10^4 CFU / mL of E. coli O157 in LB and incubated at 37°C for 16 hours. Lactea lactoferrin (Lactea-Lf) was produced as described in the above examples. Three independent lots of Lactea-Lf were assessed: a non-pure sample produced by co-elution of lactoperoxidase and lactoferrin (co-elution), a sample with degradation products produced by eluting lactoferrin from old milk left for 3 days (degradation products), and Sigma Aldrich lactoferrin L9507 (study baseline). As shown in Figure 16 and quantified in Table 5, the assessed Lactea-Lf lots showed less than 5% total bacterial growth compared to the growth of untreated bacteria under these conditions, as assessed by OD600. In this case, the co-eluting sample was one that had a detectable peak of LPO observable by MALDI-TOF mass spectrometry or HPLC. The degradation product had an identifiable degradation peak observed by HPLC or DSC. In contrast, both "non-pure" samples containing intentionally produced LPO or partially degraded lactoferrin samples showed a significant reduction in antimicrobial activity, ranging from 25–75% bacterial growth compared to the untreated control. Notably, the study standard Sigma Aldrich lactoferrin exhibited less activity than any lot of the co-eluting or degraded products.

[0210] For a more detailed comparison of the antimicrobial activity of Lactea-Lf compared to Sigma Aldrich lactoferrin, Lactea-Lf and Sigma Aldrich lactoferrin were incubated at 1 mg / mL with E. coli O157 in LB seeded at 10^4 CFU / mL. As shown in Figure 17 and quantified in Table 6, Lactea-Lf demonstrated almost complete inhibition of bacterial growth up to 18 hours, after which growth became observable at 21 hours, although bacterial growth under study standards was evident after 6 hours of growth. Results were quantified and reported as a percentage of OD600 of the untreated control at 24 hours for all time points. [Table 5-2] [Table 6]

[0211] Example 11. Assessment of lactoferrin biological activity by growth inhibition. Lactoferrin was assessed for its inhibitory effect on biofilm growth. To study the relative concentration-dependent efficacy of Lactea-Lf and Sigma Aldrich reference lactoferrin in inhibiting biofilm formation, Lf was added to E. coli O157 in LB seeded at 10^4 CFU / mL at concentrations ranging from 2 mg / mL to 0.31 mg / mL (2x serial dilution curve) and incubated at 37°C for 24 hours. Lactea lactoferrin (Lactea-Lf) was produced as described in the examples above. Biofilms were stained using a standard crystal violet assay. Quantification of biofilm formation inhibition (Figure 18A) and images of representative plates (Figure 18B) are shown. The quantification is shown in Table 7. While Lactea-Lf exhibits strong biofilm inhibition at concentrations above 100 μg / mL, Sigma Lf appears to inhibit biofilm only in the low mg / mL range, and does not completely eliminate biofilm formation.

[0212] Suspension growth in bacterial and fungal cultures was assessed for all listed species using 10-fold serial dilutions of Lf. Each listed species was grown under its own standard laboratory conditions. Lf was considered to have a strong effect on suspension growth if the OD600 was reduced by at least 90% severely at a concentration of 10 mg / mL or less by 18 hours. Lf was considered to have some effect on total bacterial growth if it had an inhibitory effect of reducing the OD600 of suspension growth by 90% or more at a concentration of 10 mg / mL or more after 18 hours. If no inhibition of growth was observed at any concentration, Lf was considered to have no effect on suspension growth.

[0213] Regarding biofilms, in the crystal violet assay, Lf was considered to have strong activity if the biofilm was eliminated at a concentration of 10 mg / mL or less after 24 hours of growth, or significantly reduced to less than 90% of the control group's level. Bacteria that did not form biofilms under any of the tested conditions and for which biofilm elimination could not be tested are indicated as N / A.

[0214] As summarized in Table 8, Lf potently inhibited both total bacterial growth and biofilm formation for all Gram-negative bacteria and yeasts tested. However, for Gram-positive bacteria, Lf generally inhibited biofilm formation rather than total bacterial growth. [Table 7] [Table 8]

[0215] Example 12. Assessment of lactoferrin wound healing. Lactea lactoferrin (Lactea-Lf) was produced as described in the above examples and freeze-dried into a powder. Lactea-Lf was then administered as a freeze-dried powder to animals with various wound conditions.

[0216] In the first assessment of the mouse model setup, mice were anesthetized with isoflurane, and two 10 mm puncture wounds were created on the back of each mouse through the skin. Three of the mice were treated with Lf by directly adding lyophilized powder to the wound bed while still anesthetized immediately after wound creation, while the other three mice were left completely untreated. The mice were then moved to non-sterile containment and observed daily. As shown in Figure 19, by day 12, all three untreated mice had died, while in the Lf-treated group, only one mouse died on day 14, and the other two survived until the end of the study on day 28. Notably, no toxicity was observed when pure lyophilized Lf powder was administered to the wounds.

[0217] In the first assessment of the pig model setting, six wounds were created in each pig using a dermatome, three on the left lateral side and three on the right lateral side, covering a surface area of ​​40 × 40 mm with a depth of 0.76 mm. Figure 20 (leftmost panel) illustrates how the wounds appeared after initial creation. For each pig, the wounds on the left side of the animal were treated, while the larger wounds on the right side were left as an untreated control. In this study, three pigs were treated with either Lactea-Lf (Figure 20; second from the left panel), administered to coat the exposed wound bed as a powder directed into the wound bed, or standard medical treatments such as bacitracin (Figure 20; second from the right panel) or silver sulfadiazine cream (Figure 20; rightmost panel). Standard medical treatments were recommended by veterinarians with experience in wound management in farm settings. As shown in Figure 20 (second from the left panel), in pigs treated with Lactea-Lf, new skin was clearly and distinctly present at the first daily observation, indicating that healing occurred within one day. However, in the standard medical control group (two panels on the right), the treated wounds were still open after one week.

[0218] In another case study, a cow with a large, infected chronic wound on its leg, which had proven resistant to other antibiotics, was treated with Lactea-Lf three times a day, applied directly into the wound bed as a freeze-dried powder. For these daily treatments, Lf was applied once each day to cover the open wound area. As shown in Figure 21, the wound had been open and expanding for several months, but after Lactea-Lf administration, the wound was completely healed after a check-up three weeks later, and no further intervention was performed after these three initial treatments.

[0219] Example 13. Further assessment of lactoferrin purity. Additional batches of Lactea-Lf described in the above examples and the relative ratio of apolactoferrin to hololactoferrin were assessed by differential scanning calorimetry (DSC). Lactoferrin samples were filled into NanoDSCs (TI Instruments) at 5 mg / mL and measured in the range of 30°C to 100°C. As shown in Figure 22, Lactea-Lf has two denatured peaks, the first corresponding to apo-Lf at approximately 60°C and the second corresponding to holo-Lf at 90°C. The ratio between these peaks is strongly skewed towards apo-Lf, but generally apo-Lf is judged to be the native form. However, two separate lots of Sigma Aldrich lactoferrin L9507 (study standard) showed an unexpected pattern; one lot was almost entirely holo-Lf, with two peaks in the holo-Lf range, which was not observed in the other. A second batch of the same product had two faint peaks, with slightly more holo-Lf than apo-Lf. These peaks may be smaller when the sample is highly impure or contains already denatured product. In either case, the presence of apo-Lf (generally judged to be necessary for the ability to exhibit chelating activity) is small as a percentage of total protein relative to study-referenced Lf.

[0220] Example 14. Further assessment of lactoferrin binding activity. The macromolecular binding characteristics of lactoferrin were evaluated using isothermal titration calorimetry (ITC). Generally, the idea that binding to heparan sulfate proteoglycans (HSPGs) is important for lactoferrin's antiviral activity is well established (Lang et al., “Inhibition of SARS pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans” PLoS One. 2011;6(8):e23710. Epub Aug 22. 2011). Additionally, binding to lipopolysaccharides (LPS) is generally considered important for lactoferrin's particularly strong activity against LPS-expressing Gram-negative bacteria. Representative ITCs comparing the binding of Sigma Aldrich lactoferrin L9507 (study standard) and Lactea-Lf are shown in Figure 23A. For this assay, Lf was prepared at 5 mg / mL in 100 mM MOPS buffer (pH 7). Heparan sulfate (HS) (Selleck Chem, Houston, TX, USA) was prepared at a concentration of approximately 100 μM in the same buffer as Lf (the concentration is approximate because the molecular weight of HS is in the range of 15-30 kDa). Lactea-Lf exhibits much higher binding to heparan sulfate than the study standard. Additionally, Figure 23B shows that Lactea-Lf also binds to LPS using a similar ITC-based assay (ThermoFisher 00-4976-93). This binding to LPS competes with HS binding. This is consistent with previous assumptions that LPS contamination, which may be present in other lactoferrin preparations, reduces subsequent binding ability and, consequently, the antimicrobial and antiviral activity of lactoferrin. The strong binding ability of Lactea-Lf demonstrates that Lactea-Lf is free from LPS contamination from raw materials that are not removed during purification.

[0221] Equal portions While the present invention has been particularly shown and described with respect to preferred embodiments and various alternative embodiments, it will be understood by those skilled in the art that various modifications in form and detail can be made therein without departing from the spirit and scope of the invention.

[0222] Reference All references, granted patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. A pharmaceutical composition comprising lactoferrin, wherein the pharmaceutical composition is formulated for delivery to a non-human subject.

2. The pharmaceutical composition according to claim 1, wherein the non-human subject is a livestock animal.

3. The pharmaceutical composition according to claim 2, wherein the livestock animal is a horse, a cow, a pig, a sheep, a goat, a chicken, or a camel.

4. The pharmaceutical composition according to claim 1, wherein the non-human subject is a pet.

5. The pharmaceutical composition according to claim 4, wherein the pet is a cat or a dog.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is formulated as animal feed, animal water, animal chew, pet food additive, or animal milk substitute.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is formulated for topical administration.

8. The pharmaceutical composition according to claim 7, wherein the formulation for local administration includes a powder.

9. The pharmaceutical composition according to claim 8, wherein the powder includes freeze-dried powder.

10. The pharmaceutical composition according to claim 7, wherein the formulation for local administration comprises microbeads.

11. The pharmaceutical composition according to claim 10, wherein the microbeads include microbeads that have been dried in a fluid bed.

12. The pharmaceutical composition according to claim 11, wherein the fluidized bed-dried microbeads are produced by granulation, encapsulation, coating, or aggregation.

13. The pharmaceutical composition according to claim 7, wherein the formulation for local administration comprises Teat Dip.

14. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is formulated as a nasal spray.

15. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is formulated for injection into the non-human animal.

16. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is formulated in an inhalation format.

17. The pharmaceutical composition according to claim 16, wherein the inhalation format is a nebulizer, inhaler, aerosol, or spray format.

18. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is formulated for delivery to a wound.

19. The pharmaceutical composition according to claim 18, wherein the pharmaceutical composition is formulated for delivery to skin tissue, eye tissue, connective tissue, mucous membrane tissue, gastrointestinal tissue and / or lung tissue.

20. The pharmaceutical composition according to claim 18, wherein the wound includes an open wound or an ulcer.

21. The pharmaceutical composition according to claim 20, wherein the ulcer is venous or arterial.

22. The pharmaceutical composition according to any one of claims 18 to 21, wherein the wound is selected from the group consisting of burns, bites, cuts, infections, ulcer formation, and combinations thereof.

23. The pharmaceutical composition according to claim 22, wherein the infection is mastitis.

24. The pharmaceutical composition according to claim 22, wherein the burn is selected from the group consisting of thermal burns, chemical burns, electrical burns, radiation burns, friction burns, and combinations thereof.

25. The pharmaceutical composition according to claim 22, wherein the cut is selected from the group consisting of puncture wounds, lacerations, abrasions, incisions, severances, and combinations thereof.

26. The pharmaceutical composition according to claim 25, wherein the incision includes a surgical incision.

27. The pharmaceutical composition according to any one of claims 18 to 26, wherein the pharmaceutical composition comprises a bioadhesive.

28. The pharmaceutical composition according to any one of claims 18 to 27, wherein the pharmaceutical composition is formulated in a delivery format selected from the group consisting of gel foam, hydrogel, injected bandage, powder, spray, and inhalation format.

29. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is formulated for delivery to the gastrointestinal tract.

30. The pharmaceutical composition according to claim 29, wherein the pharmaceutical composition is formulated in a delivery format including microencapsulation, microbeads, or an inert substance containing lactoferrin.

31. The pharmaceutical composition according to claim 30, wherein the lactoferrin is produced via fluidized bed drying to generate the powder or microbeads via granulation, encapsulation, coating, or aggregation.

32. The pharmaceutical composition according to claim 30 or 31, wherein the pharmaceutical composition is formulated in a delivery format including an enteric coating.

33. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is formulated for dental administration.

34. The pharmaceutical composition according to claim 33, wherein the preparation for dental administration is an oral spray, a vegetarian chew, or a pet food additive.

35. A pharmaceutical composition comprising lactoferrin, which is formulated for dental administration.

36. The pharmaceutical composition according to claim 33, wherein the preparation for dental administration is toothpaste, mouthwash, floss, oral spray, effervescent tablet, mouth prop, retainer, oral packing material, or dental instrument disinfectant.

37. A pharmaceutical composition comprising lactoferrin, which is formulated for delivery to the eye.

38. The pharmaceutical composition according to claim 37, wherein the formulation for delivery to the eye comprises an eye drop solution, a contact lens solution, an eye wash solution, or an eye spray.

39. A composition comprising lactoferrin, wherein the composition is configured in a reproductive health format.

40. The composition according to claim 39, wherein the reproductive health format comprises a lubricant, a condom, a gel, an injected wipe, or an injected pad.

41. A composition comprising lactoferrin, which is formulated for disinfection of a surface.

42. The composition according to claim 41, wherein the surface includes a medical surface, a hospital instrument, a surgical instrument, a surgical implant, a dental instrument, a food preparation surface, skin, a nipple, or a flesh surface.

43. A composition comprising lactoferrin, which is formulated for use as a food or in a food product.

44. The composition according to claim 43, wherein the food and / or food product comprises animal feed, milk substitute, colostrum substitute, water, beverage, or pre-packaged food.

45. A composition comprising lactoferrin, which is formulated as a cosmetic.

46. The composition according to claim 45, wherein the cosmetic comprises a cream, lotion, sunscreen, or spray.

47. Any one claim of a prior pharmaceutical composition or composition, wherein the pharmaceutical composition or composition comprises an excipient.

48. Any one claim of a prior pharmaceutical composition or composition, wherein the pharmaceutical composition or composition comprises sodium stearyl fumarate, carbomer, sodium hydroxide, glycerin, sodium benzoate, benzoic acid, citric acid, and / or a combination thereof.

49. Any one claim of a prior pharmaceutical composition or composition, wherein the pharmaceutical composition or composition comprises xylitol, sorbitol, mannitol, maltitol, sugar alcohol, sucrose, and / or a combination thereof.

50. Any one claim of a prior pharmaceutical composition or composition, wherein the pharmaceutical composition or composition comprises isomalt, microcrystalline cellulose, sodium carboxymethylcellulose, and / or a combination thereof.

51. Any one claim of a prior pharmaceutical composition or composition, wherein the pharmaceutical composition or composition comprises powdered sugar, gum base, corn syrup, gum arabic, and / or a combination thereof.

52. Any one claim of a prior pharmaceutical composition or composition, wherein the pharmaceutical composition or composition comprises HiG PWD-04, encapsulated powder flavoring substance, anhydrous caffeine or natural caffeine, encapsulated sucralose or sucralose, encapsulated acesulfame K and encapsulated aspartame, aspartame, acesulfame K, triacetin, silicon dioxide, and / or a combination thereof.

53. Any one claim of a prior pharmaceutical composition or composition, wherein the pharmaceutical composition or composition comprises a coagulating substance.

54. The pharmaceutical composition or composition according to claim 53, wherein the coagulation substance comprises thrombin, amylopectin, kaolin, and / or a combination thereof.

55. The pharmaceutical composition or composition according to any one of the prior claims, wherein the pharmaceutical composition or composition comprises an astringent substance.

56. The pharmaceutical composition or composition according to claim 55, wherein the astringent substance 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.

57. The pharmaceutical composition or composition according to any one of the prior claims, wherein the lactoferrin is bovine.

58. The pharmaceutical composition or composition according to any one of the prior claims, wherein the lactoferrin is untreated.

59. The pharmaceutical composition or composition according to any one of the prior claims, wherein the lactoferrin has not been chemically treated, enzymatically treated, acidically treated, and / or heat-treated.

60. The pharmaceutical composition or composition according to any one of the prior claims, wherein the lactoferrin has not been heat-treated.

61. The pharmaceutical composition or composition according to any one of claims 58 to 60, wherein 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.

62. The pharmaceutical composition or composition according to any one of claims 58 to 60, wherein the lactoferrin has not been heat-treated at a temperature of 55°C or higher.

63. The pharmaceutical composition or composition according to any one of the prior claims, wherein the purified lactoferrin includes a native conformation assessed by circular dichroism.

64. The pharmaceutical composition or composition according to any one of the prior claims, wherein the purified lactoferrin includes a native conformation determined by differential scanning calorimetry (DSC).

65. The pharmaceutical composition or composition according to claim 64, wherein the native conformation includes the apolactoferrin conformation and / or the hololactoferrin conformation.

66. The pharmaceutical composition or composition according to claim 65, wherein the apolactoferrin conformation has a melting temperature peak at 60.2 ± 8°C, and / or the hololactoferrin conformation has a melting temperature peak at 88.38 ± 8°C.

67. The pharmaceutical composition or composition according to any one of the prior claims, wherein the purified lactoferrin is capable of binding iron.

68. The pharmaceutical composition or composition according to claim 67, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the purified lactoferrin is capable of binding iron.

69. The pharmaceutical composition or composition according to claim 67, wherein at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the purified lactoferrin is capable of binding iron.

70. The pharmaceutical composition or composition according to claim 67, wherein 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.

71. A pharmaceutical composition or composition according to any one of claims 67 to 70, wherein the ability to bind the iron is assessed by DSC.

72. The pharmaceutical composition or composition according to any one of the prior claims, wherein the purified lactoferrin includes post-translational modifications.

73. The pharmaceutical composition or composition according to claim 72, wherein the post-translational modification includes glycosylation.

74. The pharmaceutical composition or composition according to any one of the prior claims, wherein the purified lactoferrin contains an average molecular weight of at least 79,000 to 86,000 Da.

75. The pharmaceutical composition or composition according to any one of the prior claims, wherein the purified lactoferrin is dried.

76. The pharmaceutical composition or composition according to claim 75, wherein the purified lactoferrin is dried by freeze-drying, fluidized bed drying, or low-temperature spray drying.

77. The pharmaceutical composition or composition according to any one of the prior claims, wherein the purified lactoferrin remains in liquid form after lactoferrin purification.

78. The pharmaceutical composition or composition according to any one of the prior claims, wherein the composition further comprises iron molecules.

79. The pharmaceutical composition or composition according to any one of the prior claims, wherein the purified lactoferrin is complexed with an iron molecule.

80. The aforementioned iron molecule, Fe ２＋ or Fe ３＋ A pharmaceutical composition or composition according to claim 78 or 79, comprising:

81. The pharmaceutical composition or composition according to any one of the prior claims, wherein the purified lactoferrin is complexed with a copper molecule, a zinc molecule, a manganese molecule, and / or a gallium molecule.

82. The pharmaceutical composition or composition according to any one of the prior claims, wherein the purified lactoferrin is complexed with a zinc molecule.

83. The pharmaceutical composition or composition according to any one of the prior claims, wherein the composition contains endotoxin at a level of 5 EU / kg or less.

84. The pharmaceutical composition or composition according to any one of the prior claims, wherein the lactoferrin is purified from an untreated natural dairy product.

85. The pharmaceutical composition or composition according to claim 84, wherein the natural dairy product has not been processed before the purification of the lactoferrin.

86. The pharmaceutical composition or composition according to claim 84, wherein the natural dairy product has not been chemically treated, enzymatically treated, acidically treated, or heat-treated before the purification of the lactoferrin.

87. The pharmaceutical composition or composition according to claim 84, wherein the natural dairy product has not been heat-treated before the purification of the lactoferrin.

88. The pharmaceutical composition or composition according to any one of claims 86 or 87, wherein the heat treatment includes 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.

89. The pharmaceutical composition or composition according to claim 86 or 87, wherein the heat treatment includes a temperature of 55°C or higher.

90. The pharmaceutical composition or composition according to any one of the prior claims, wherein the lactoferrin is purified from natural dairy products separated into skim milk and cream before the purification of the lactoferrin.

91. The pharmaceutical composition or composition according to claim 90, wherein the separation into skim milk and cream includes cold bowl separation.

92. The pharmaceutical composition or composition according to any one of the prior claims, wherein the lactoferrin is purified from a natural dairy product that has been acid-treated before the purification of the lactoferrin.

93. The pharmaceutical composition or composition according to claim 92, wherein the acid treatment includes the removal of insoluble casein.

94. The pharmaceutical composition or composition according to claim 92 or 93, wherein the acid treatment is performed at a pH of 4.0 or higher.

95. The pharmaceutical composition or composition according to any one of the prior claims, wherein the lactoferrin is present in a concentration of 0.1 to 0.5 mg / ml, 0.125 to 0.5 mg / ml, 0.1 to 1 mg / ml, 0.1 to 0.25 mg / ml, or 0.125 to 0.25 mg / ml.

96. The pharmaceutical composition or composition according to any one of the prior claims, wherein the lactoferrin is present at a concentration of 0.125 to 0.5 mg / ml.

97. The pharmaceutical composition or composition according to any one of the prior claims, wherein the lactoferrin is present in a concentration of 1 mg / ml or less.

98. A method for producing a prior pharmaceutical composition or one of the compositions.

99. The method according to claim 98, wherein the method comprises one or more steps selected from the group consisting of chromatography, filtration, and drying.

100. The method according to claim 98, wherein the method comprises each of the steps of chromatography, filtration, and drying.

101. The method according to any one of claims 98 to 100, wherein the method includes pressing into a drug dispensing form.

102. A method for treating a disease or condition, comprising administering a prior pharmaceutical composition or one of the compositions.

103. The method according to claim 102, wherein the disease or condition is selected from the group consisting of pathogenic diseases, gastrointestinal (GI) infections, wound infections, allergic conditions, inflammatory conditions, and combinations thereof.

104. The method according to claim 102, wherein the disease or condition is mastitis.

105. A method for treating a pathogenic disease, comprising administering a prior pharmaceutical composition or one of the compositions to a subject.

106. The method according to any one of claims 102 to 105, wherein the administration is prophylactic.

107. The method according to claim 106, wherein the subject is at risk of exposure to a pathogen.

108. The method according to claim 106, wherein the subject is exposed to a pathogen.

109. The method according to claim 105, wherein the subject has been diagnosed with an infection caused by a pathogen.

110. A method for reducing the risk of pathogenic disease, comprising administering a prior pharmaceutical composition or one of the compositions to a subject.

111. The method according to claim 110, wherein the reduction of the risk of pathogenic disease includes administering the pharmaceutical composition or the composition to the target wound.

112. The method according to claim 111, wherein the pharmaceutical composition or the administration of the composition includes local administration.

113. The method according to claim 112, wherein the local administration comprises the administration of a powder containing lactoferrin, microbeads, or an inactive substance.

114. The method according to claim 113, wherein the lactoferrin comprises fluidized bed dried lactoferrin.

115. The method according to claim 113 or 114, wherein the lactoferrin is produced via fluidized bed drying to generate the powder or microbeads via granulation, encapsulation, coating, or aggregation.

116. The method according to claim 113, wherein the powder includes freeze-dried powder.

117. The method according to any one of claims 110 to 116, wherein the subject is exposed to a pathogen and / or has been diagnosed with an infection by the pathogen.

118. The method according to any one of claims 103 to 117, wherein the pathogen comprises a microorganism that is transmitted orally and / or nasally.

119. The method according to any one of claims 103 to 118, wherein the pathogen includes a floating microorganism.

120. The method according to any one of claims 103 to 119, wherein the pathogen includes a virus.

121. The method according to claim 120, wherein the virus includes respiratory viruses.

122. The method according to any one of claims 107 to 119, wherein the pathogen comprises bacteria or fungi.

123. The method according to any one of claims 102 to 122, wherein the treatment of the disease or condition includes preventive treatment of the disease or condition.

124. A method for reducing the risk of infection, comprising administering one of the preceding pharmaceutical compositions or compositions to a subject.

125. The method according to claim 124, wherein the subject has a wound that carries a risk of infection.

126. The method according to claim 125, wherein the pharmaceutical composition or the administration of the composition includes local administration.

127. The method according to claim 126, wherein the local administration comprises the administration of a powder containing lactoferrin, microbeads, or an inactive substance.

128. The method according to claim 127, wherein the lactoferrin comprises fluidized bed dried lactoferrin.

129. The method according to claim 127 or 128, wherein the lactoferrin is produced via fluidized bed drying to generate the powder or microbeads via granulation, encapsulation, coating, or aggregation.

130. The method according to claim 127, wherein the powder comprises freeze-dried lactoferrin.

131. A method for promoting wound healing of tissue, comprising administering a prior pharmaceutical composition or one of the compositions to a wound in the target tissue.

132. The method according to claim 131, wherein the wound is selected from the group consisting of burns, cuts, infections, ulceration, and combinations thereof.

133. The method according to claim 132, wherein the burn is selected from the group consisting of thermal burns, chemical burns, electrical burns, radiation burns, friction burns, and combinations thereof.

134. The method according to claim 132, wherein the cut is selected from the group consisting of puncture wounds, lacerations, abrasions, incisions, severances, and combinations thereof.

135. The method according to claim 134, wherein the incision is a surgical incision.

136. The method according to any one of claims 131 to 135, wherein the tissue is skin, lung tissue, or a combination thereof.

137. The method according to any one of claims 131 to 136, wherein the method comprises treating a disease or condition selected from the group consisting of pathogenic diseases, gastrointestinal (GI) infections, wound infections, allergic conditions, inflammatory conditions, and combinations thereof.

138. The method according to claim 137, wherein the treatment of the disease or condition comprises reducing pain and / or inflammation.

139. The method according to claim 137 or 138, wherein the treatment of the disease or condition includes preventive treatment of the disease or condition.

140. The method according to any one of claims 131 to 138, wherein the pharmaceutical composition or the administration of the composition includes local administration to the wound.

141. The method according to claim 140, wherein the local administration includes the administration of a powder.

142. The method according to claim 141, wherein the powder includes freeze-dried powder.

143. A method for disinfecting a surface, comprising applying a prior pharmaceutical composition or one of the compositions to the surface.

144. The method according to claim 143, wherein the surface includes a medical surface, a hospital instrument, a surgical instrument, a surgical implant, a dental instrument, a food preparation surface, skin, a nipple, or a flesh surface.

145. The method according to claim 143 or 144, wherein the skin includes a wound.

146. The method according to any one of claims 143 to 145, wherein the disinfection is capable of inhibiting microbial growth.

147. The method according to claim 146, wherein the inhibition of microbial growth includes inhibiting or preventing biofilm formation.

148. The method according to any one of claims 143 to 147, wherein the lactoferrin is concentrated at a concentration of 0.1 to 0.5 mg / ml, 0.125 to 0.5 mg / ml, 0.1 to 1 mg / ml, 0.1 to 0.25 mg / ml, or 0.125 to 0.25 mg / ml.

149. The method according to any one of claims 143 to 147, wherein the lactoferrin is present at a concentration of 0.125 to 0.5 mg / ml.

150. The method according to any one of claims 143 to 147, wherein the lactoferrin is at a concentration of 1 mg / ml or less.

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