Method for producing lactoferrin powder and uses thereof

Abstract

Disclosed herein is a process for producing lactoferrin powder. The process includes extracting lactoferrin from a natural lactoferrin-containing material and concentrating the extracted lactoferrin to produce a liquid concentrate with improved stability of the extracted lactoferrin. The liquid concentrate is then pasteurized by heating to a temperature and for a time effective to pasteurize, and the pasteurized liquid concentrate is then dried under non-denaturing conditions to produce a lactoferrin powder in which substantially all of the lactoferrin is natural lactoferrin.

Description

[Technical field]

[0001] Methods for producing lactoferrin powders and uses of such powders are described herein. [Background technology]

[0002] Lactoferrin is a protein found in many animal secretions, including animal milk. It is believed to have many health effects or benefits and is an ingredient in many milk replacer formulations, such as infant formulas.

[0003] Lactoferrin can be produced recombinantly or by extraction from other dairy products such as milk or cheese whey. A typical process for producing lactoferrin from milk or cheese whey involves an initial pasteurization step to reduce microbial load, followed by concentration and extraction of lactoferrin from the milk / whey matrix. The lactoferrin concentrate is then typically freeze-dried to produce a particulate material that is primarily composed of lactoferrin. If lactoferrin with a specific particle size and / or particle size distribution is required, the freeze-dried particles can be milled.

[0004] It would be advantageous to provide alternative methods for producing lactoferrin. Summary of the Invention

[0005] In a first aspect, the present invention provides a process for producing a lactoferrin powder, the process comprising: extracting lactoferrin from a natural lactoferrin-containing material and concentrating the extracted lactoferrin to produce a liquid concentrate in which the extracted lactoferrin has improved stability (i.e., compared to when it was in the lactoferrin-containing material); heating the liquid concentrate to a temperature and for a time effective to pasteurize the liquid concentrate; and drying the pasteurized liquid concentrate under non-denaturing conditions to produce a lactoferrin powder in which substantially all of the lactoferrin is native lactoferrin.

[0006] The present inventors, the subject of this application, recognize that conventional processes for producing lactoferrin from milk or cheese whey include steps in which the conditions experienced by lactoferrin result in some degree of degradation / denaturation of its protein structure. This causes a consequential reduction in the amount of lactoferrin recovered and / or the quality of the lactoferrin-containing product in terms of the percentage of native lactoferrin in the product. As will be appreciated, denatured lactoferrin will likely have reduced functionality and biological activity compared to native lactoferrin. Such a reduction in quality went unrecognized in the art for some time, since the techniques used to characterize lactoferrin-containing products did not always sufficiently distinguish between native and non-native forms of lactoferrin, and the products contained enough native lactoferrin to have some level of functionality. In the present invention, more lactoferrin can be recovered than in many conventional processes, and the recovered lactoferrin is less denatured than in many cases. In effect, the lactoferrin in the lactoferrin powder of the present invention retains most of its biological activity, despite being presented in a processed and pasteurized form.

[0007] Lactoferrin, like many proteins, is heat sensitive and has been found to be particularly susceptible to degradation when exposed to pH changes at high temperatures, as can occur when liquids such as milk, whey, and the like are heated (particularly above pasteurization temperatures). Furthermore, other components of the liquids in which lactoferrin is typically provided have been found to contribute to the destabilization of lactoferrin and render it more susceptible to degradation when heated. The inventors have discovered that when lactoferrin presented at the pasteurization stage (specifically, although it is understood that heat may be applied at other stages of the process) is extracted from an unpasteurized milk matrix into a liquid concentrate with improved stability of lactoferrin (i.e., when compared to its stability in a native lactoferrin-containing material, such as milk), a lactoferrin-containing powder can be produced in which substantially all of the lactoferrin (as defined below) is native (i.e., undenatured) lactoferrin.

[0008] In this way, lactoferrin is not exposed to such destabilizing effects that may occur when liquids such as milk are heated during pasteurization (which is essential for regulatory compliance with many industrial standards for food), and the process of the present invention is found to cause less damage to the structure of lactoferrin protein as in conventional processes.Indeed, the inventors observe that pasteurization of milk results in a loss of lactoferrin recovery of about 25%, and there may be some other conformational changes in lactoferrin when pasteurized in milk matrix.Furthermore, in the present invention, lactoferrin-containing powder is produced with a minimum number of process steps, and does not include steps that are normally used to produce lactoferrin powder, such as evaporation.The inventors recognize that all additional process steps may damage lactoferrin and / or result in a lower overall recovery rate.

[0009] In some embodiments, the extracted lactoferrin may have improved stability due to the absence of lactoferrin destabilizing species in the liquid concentrate.Species typically found in milk matrix include, for example, proteins such as casein and whey protein, lipopolysaccharides, sugars, and ionic species such as calcium and magnesium ions, some or all of which may adversely affect the stability of lactoferrin in milk matrix (e.g., by causing pH shift).Pasteurizing the lactoferrin-containing liquid concentrate that does not contain such potentially harmful species means that the lactoferrin is not heated in the presence of species that may have a destabilizing effect on lactoferrin.

[0010] In some embodiments, the extracted lactoferrin may have improved stability due to the presence in the liquid concentrate of species that stabilize lactoferrin. The liquid concentrate may include, for example, one or more of the following: pH adjusters, stabilizing minerals, and dissolved gases. Specific examples of such species, and the inventors' understanding of their protective mechanisms, are described below.

[0011] In some embodiments, the natural lactoferrin-containing material can be milk, preferably skim milk. It has been found that low-temperature bowl fat separation is advantageous for producing skim milk because it does not involve subjecting lactoferrin to even mildly high temperatures while still contained within the milk matrix. As described in more detail below, lactoferrin can be easily denatured in this initial step.

[0012] In some embodiments, lactoferrin may be extracted from a natural lactoferrin-containing material using ion exchange chromatography: lactoferrin may be eluted from an ion exchange column separately from other components of the material (e.g., the milk matrix).

[0013] In some embodiments, the extracted lactoferrin may be concentrated using ultrafiltration, with the lactoferrin contained in the filtrate for subsequent reconstitution in a liquid concentrate.

[0014] In some embodiments, the pasteurized liquid concentrate may be cooled immediately after pasteurization, thereby reducing the amount of time the lactoferrin spends at elevated temperatures to an absolute minimum (while still complying with regulatory requirements regarding pasteurization).

[0015] In some embodiments, the pasteurized liquid concentrate can be dried under non-denaturing conditions to produce lactoferrin-containing products by spray drying. Spray drying (especially multi-stage spray drying) has been found to be advantageous by the inventors to reduce the need for steps such as evaporation, which entail the application of potentially harmful amounts of heat, before further heat application and / or processing during the final powder formation stage. Particular advantages can be achieved when multi-stage spray drying is used, where parameters such as nozzle size, inlet and outlet temperatures, and residence time are advantageously used to further reduce the cumulative application temperature and thus contribute to producing substantially natural lactoferrin-containing powders with specific characteristics, such as specific desired particle size and / or particle size distribution.

[0016] In some embodiments, the lactoferrin powder may consist essentially of lactoferrin. In alternative embodiments, up to about 10% w / w (preferably less than about 5% w / w) of other substances may be present in the lactoferrin powder, and as such are unavoidable by-products of the process of the present invention. For example, moisture and other non-specific proteins may be included in the final product, provided that their presence does not adversely affect the usefulness of the product.

[0017] In a second aspect, the present invention provides a lactoferrin powder produced by the process of the first aspect of the invention. In a third aspect, the present invention provides a food product comprising or consisting essentially of lactoferrin powder produced by the process of the first aspect of the invention.

[0018] The inventors have also discovered that the highly functional and biologically active lactoferrin contained in the lactoferrin powder produced according to the process of the first aspect of the present invention may have therapeutic activity or enhanced therapeutic activity due to the unique properties of lactoferrin powder.In particular, the experiments carried out by the inventors (described below) have demonstrated that the powdered lactoferrin produced according to the present invention has an inhibitory effect on viral replication in vitro and in vivo.The inventors believe that the results of their preliminary experiments lead to a reasonable prediction of the therapeutic use disclosed herein.Further experiments, both currently in progress and planned, are expected to confirm the inventors' prediction.

[0019] In a fourth aspect, the present invention therefore provides a nasal spray comprising lactoferrin powder produced by the process of the first aspect of the invention. In a fifth aspect, the present invention provides a pharmaceutical composition comprising lactoferrin powder produced by the process of the first aspect of the invention and a pharma- ceutically acceptable excipient.

[0020] In a sixth aspect, the present invention provides a method for preventing or treating a viral infection in a patient, the method comprising administering to the patient (e.g. via the patient's respiratory tract) a formulation comprising lactoferrin powder produced by the process of the first aspect of the invention or the pharmaceutical composition of the fifth aspect of the invention.

[0021] In a seventh aspect, the present invention provides a method for preventing or treating a viral infection in a patient, the method comprising intranasally administering to the patient a formulation comprising lactoferrin powder produced by the process of the first aspect of the invention or the pharmaceutical composition of the fifth aspect of the invention.

[0022] In an eighth aspect, the present invention provides a lactoferrin powder produced by the process of the first aspect of the invention for use as a medicament. In a ninth aspect, the present invention provides a lactoferrin powder produced by the process of the first aspect of the invention for use in preventing or treating a viral infection in a patient.

[0023] In a tenth aspect, the present invention provides the use of lactoferrin powder produced by the process of the first aspect of the invention for the manufacture of a medicament for preventing or treating a viral infection in a patient.

[0024] The inventors also note that lactoferrin has demonstrated immunomodulatory and anti-inflammatory activity, and that the lactoferrin powder produced by the process of the first aspect of the invention may provide an improved delivery form of lactoferrin for the treatment and prevention of conditions associated with such activity.

[0025] Other aspects, features, and advantages of the present invention are described below. [Brief description of the drawings]

[0026] Embodiments of the invention are described in further detail below with reference to the following drawings: [Figure 1] FIG. 2 is a block flow diagram of an embodiment of the first aspect of the present invention. [Diagram 2] Graph showing viral load from a preliminary BEC study using cells from an asthma patient, n=1. Differentiated primary human bronchial epithelial cells with OC43 viral load after treatment with 100ug / mL (LF100■) or 1000ug / mL (LF1000▲). [Diagram 3] Graph showing viral load from another pilot study using n=2 healthy donors (n=4 each replicates=8 total), untreated (●) and 1000ug / mL (LF1000▲). [Figure 4]Graph showing the number of infectious viruses over time for 2× healthy donors (n=4 each replicates=8 total), untreated (●), 100ug / mL (LF100■), and 1000ug / mL (LF1000▲). [Diagram 5] Graph showing the number of infectious viruses over time for n=1 healthy donor (n=3 each replicates=6 total, lactoferrin supplemented at 48 hours), untreated (●), and 100ug / mL (LF1000■). [Figure 6] (A) Experimental design for in vivo studies 1 and 2, and (B) in vivo study 3 are shown. [Figure 7] Figure 7A is a graph showing OC43 viral load in nasal turbinates in untreated (vehicle) mice versus mice treated intranasally with 1.5ug lactoferrin over a 48 hour time course. Figure 7B is a graph showing OC43 infectious virus measured in MRC-5 cells using a TCID50 assay in nasal wash samples. [Figure 8] Figure 8A is a graph showing OC43 viral load in nasal turbinates in untreated (vehicle) mice versus mice treated intranasally with 1.5ug lactoferrin over a 48 hour time course. Figure 8B is a graph showing OC43 infectious virus measured in MRC-5 cells using a TCID50 assay in nasal wash samples. [Figure 9] Graph showing A) IFN-β and B) IFN-λ2 / 3 levels between vehicle and lactoferrin treated mice as assessed by qPCR. [Figure 10] Graphs showing (A) macrophages, (B) neutrophils, and (C) lymphocytes in the nasal turbinates in untreated (vehicle) mice versus mice treated with 150 ug lactoferrin at the time of infection and twice daily thereafter as well as BAL. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] As described above, the present invention provides a process for producing a lactoferrin powder, which the inventors believe to have unique properties in that the lactoferrin in the powder composition retains most of its biological activity despite being pasteurized and processed into a powder. The process involves extracting lactoferrin from a natural lactoferrin-containing material and concentrating the extracted lactoferrin to produce a liquid concentrate having improved stability (as evidenced by the lactoferrin in the powder substantially retaining its biological and functional properties, as well as its % recovery, as described below) compared to when the extracted lactoferrin was in the lactoferrin-containing material. The liquid concentrate is then pasteurized by heating to a temperature and for a time effective to pasteurize, after which the pasteurized liquid concentrate is dried under non-denaturing conditions to produce a lactoferrin powder in which substantially all of the lactoferrin is natural lactoferrin.

[0028] The lactoferrin powder so produced has improved biological activity relative to many other lactoferrin-containing powders due to its higher percentage of native lactoferrin. The functional properties of the lactoferrin powder are also advantageous (e.g., due to its solubility, blendability specific particle size attributes and morphology, etc.). The process of the present invention can also result in improved yields of native lactoferrin when compared to processes in which lactoferrin extraction is performed after pasteurization.

[0029] The present invention results in the production of lactoferrin powder in which substantially all of the lactoferrin is natural lactoferrin. Natural lactoferrin is undenatured or only negligibly altered, and lactoferrin protein retains its structure and therefore its biological activity and health effects. As will be understood, not all lactoferrin processed according to the present invention may be completely undenatured. Some denaturation may be unavoidable. Lactoferrin powder may contain, for example, more than 90% natural lactoferrin, more than 95% natural lactoferrin, or more than 98% natural lactoferrin. Lactoferrin powder may contain, for example, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% natural lactoferrin.

[0030] The lactoferrin powder produced by the present invention may consist essentially of lactoferrin (i.e., the product entity is lactoferrin, except for small amounts of unavoidable impurities, by-products, degradation products, etc.). However, such a high level of purity may not be required, and in some embodiments (e.g., where a cheaper product is commercially attractive), it may be sufficient for up to about 10% w / w of other components to be present in the lactoferrin powder. Such other components may include unavoidable by-products of the process of the present invention, but may also include additives provided for functional reasons (e.g., anti-caking agents, preservatives, etc.).

[0031] The lactoferrin powder produced by the present invention can have any suitable particle size or particle size range, which is described in more detail below in the context of the drying step.As an example, one lactoferrin-containing powder produced according to the present invention and characterized by the inventors as a fine powder has a particle size distribution D50=38.2 microns and D90=72.3 microns. Another lactoferrin-containing powder produced according to the present invention and characterized by the inventors as a medium powder has a particle size distribution D50=46.2 microns and D90=98.7 microns. Yet another lactoferrin-containing powder produced according to the present invention and characterized by the inventors as a coarse powder has a particle size distribution D50=56 microns and D90=130 microns.

[0032] The percentage of native lactoferrin in any given sample of lactoferrin-containing powder can be determined as described below in conjunction with published techniques. Generally speaking, native lactoferrin protein has a molecular weight of about 80 kDa, has about 10-20% iron saturation, and is salmon pink in color. Native lactoferrin powder has biological activity equivalent to its source, while damaged lactoferrin has impaired biological activity.

[0033] The assay that we used to determine lactoferrin purity in lactoferrin powder (e.g., percentage purity of lactoferrin versus other proteins in a sample), but do not believe to be suitable for quantification or distinguishing between native (i.e., non-denatured) and denatured forms of lactoferrin, is the Chinese GB RP-HPLC (GB 1903.17-2016) method. Implemented by the Standards Administration of China to test bovine lactoferrin powder imported into China, this method involves injection of a high concentration of protein (50 μl of 10 mg / ml sample (0.5 mg per injection)), which was close to the upper limit of the column binding capacity. Because of this, some notable differences were observed in the bovine lactoferrin determination.

[0034] The assay we used to determine the amount of lactoferrin (as a percentage) in the finished powder is the Callaghan Innovation RP-HPLC method, where only native lactoferrin binds to the HPLC column, making it distinguishable from denatured lactoferrin. The Callaghan Innovation method is described in J.Billakanti et.el., International Dairy Journal 99(2019)104546, and is performed using an Aeris™ 3.6μm WIDEPORE XB-C8 200 Å analytical LC column (Part-OOG-4481-EO, 250[1]4.6mm), C8 Security Guard Ultra Holder (Part-AJO-9000), and Security Guard Ultra Cartridges (Part-AJO-8771), with detection at a wavelength of 280nm. The method uses w / w preparation of samples rather than v / v-based sample preparation used in other protocols, which results in reproducible and reliable results over time and, as such, more accurate analysis.

[0035] The inventors have found that such a combination of assays allows both the lactoferrin quantity (i.e., percentage recovery) and quality (i.e., percentage of native lactoferrin) of a particular lactoferrin-containing powder to be determined. As noted above, the present invention, the subject matter of this application, is directed, at least in part, to the recognition that both of these assays are necessary to adequately characterize a native lactoferrin-containing powder.

[0036] One of the important features of the present invention is that pasteurization is performed only after lactoferrin is extracted from the natural lactoferrin-containing material and concentrated. The lactoferrin in the liquid concentrate has improved stability compared to when it was in the natural lactoferrin-containing material, and is therefore less susceptible to denaturation caused by the temperature that must be applied to pasteurize the product. Although pasteurization conditions (minimum temperature, time, etc.) must be met to meet regulatory (and safety) requirements, in the present invention, the stability of lactoferrin to the applied heat is greater than if pasteurization was performed while lactoferrin was contained in the natural lactoferrin-containing material. In this way, substantially all of the lactoferrin in the liquid concentrate can remain in its natural form. In contrast, pasteurization of milk, for example, can denature up to 25% of the natural lactoferrin in milk, resulting in a corresponding decrease in lactoferrin recovery at the start.

[0037] In the context of the present invention, the feature "lactoferrin with improved stability" and the like is understood to mean that the stability of lactoferrin in a liquid concentrate is greater than the stability of lactoferrin in a natural lactoferrin-containing material. Routine tests (including those described below) can be used to assess the relative stability of lactoferrin in different carriers.

[0038] Unless protected by the methods described herein, lactoferrin may undergo either or both thermal and chemical (e.g., pH shifts cause denaturation) degradation. Lactoferrin may have improved thermal stability due to the absence in the liquid concentrate of species that destabilize lactoferrin. Alternatively, or in addition, lactoferrin may have improved thermal stability due to the presence in the liquid concentrate of species that stabilize lactoferrin.

[0039] As mentioned above, species typically found in dairy products such as whey and milk include proteins such as casein and whey protein, sugars (e.g., lactose), fats (e.g., lipopolysaccharides), and ionic species such as calcium and magnesium ions. Any or all of these species may contribute to the relative lack of thermostability of lactoferrin, either directly (e.g., by denaturing or otherwise reacting with lactoferrin when heated) or indirectly (e.g., by causing a pH shift that destabilizes lactoferrin at higher temperatures). Isolating lactoferrin from the matrix of natural lactoferrin-containing material prevents (or at least reduces) the occurrence of any such adverse interactions.

[0040] The lactoferrin-containing liquid concentrate may contain additional species that can help stabilize lactoferrin when it is heated. Such species should be compatible with the end use of the lactoferrin powder (e.g., GRAS, food compatible, etc.) and not otherwise adversely affect the performance of the invention. These species may either be added to the liquid concentrate or may already be present in the concentrate (e.g., in the eluate).

[0041] In some embodiments, the liquid concentrate may include processing aids such as, for example, one or more of the following: pH adjusters, stabilizing minerals, and dissolved gases. Such species may be added to the liquid concentrate via a carrier liquid / gas or directly in their current form prior to pasteurization.

[0042] The conductivity of the liquid concentrate (which is a measure of the concentrate's ability to pass or carry electric current) can be adjusted, for example, by manipulating the amount of minerals, such as sodium chloride, or those found in town water supplies, in the concentrate. The present invention can maintain the natural aspects of lactoferrin by controlling the conductivity to further stabilize the lactoferrin concentrate before pasteurization. The liquid concentrate can be RO water, but water with a small mineral content (e.g., town water) may not adversely affect the process and can help reduce the overall cost of the process. In fact, water containing low levels of minerals can also act to further stabilize lactoferrin.

[0043] The pH of the liquid concentrate may be controlled in a manner to prevent the generation of unwanted contaminants or to meet importing country requirements or customer specifications. Generally speaking, for example, the pH of lactoferrin must be in compliance in the range of 5.2 to 7.2 in order to comply with regulatory requirements. Adjustment of the pH of the liquid concentrate may be used in two cases: firstly, to stabilize the lactoferrin for pasteurization, or secondly, to ensure final compliance with standards.

[0044] The inventors note that apo-lactoferrin (non-iron bound) is more easily damaged than holo-lactoferrin (iron bound), which is much more stable. Thus, conditions in the liquid concentrate that favor the formation of holo-lactoferrin may improve the heat resistance of lactoferrin.

[0045] The use of certain acids to reduce pH may be appropriate, as they may help to reduce the effects of denaturation.However, doing so may increase contaminant levels in the concentrate or adversely affect sensory characteristics, which is undesirable and may not be acceptable in lactoferrin-containing products in some markets.

[0046] The use of sodium bicarbonate (or other bicarbonate salts) has also been found to stabilize lactoferrin during pasteurization, presumably due to the interaction of the bicarbonate ion with the iron-binding sites on the C and N lobes of the lactoferrin molecule. Such may also help to manage the pH of the final product so that it is at a pH of 5.2-7.2 (preferably 5.7), as required for food / additives.

[0047] It is within the capabilities of one of ordinary skill in the art to use simply the information disclosed herein and routine testing and experimentation to determine whether any particular species can be used in the methods of the present invention.

[0048] As mentioned above, additional components may need to be added during the process in order to meet regulatory requirements. For example, food products are required to have a pH greater than 5.2, and in some embodiments, it may be necessary to add a pH adjuster after pasteurization in order for the end product of the present invention to be compliant. It is within the ability of one of ordinary skill in the art to determine the need for such additional components and select such additional components using the teachings contained herein as well as simply routine testing.

[0049] We also note that additional processing steps can be used instead of adding additional components to achieve the same effect, for example anion exchange can be used to adjust the pH of the sample, and it may be advantageous to perform anion exchange instead of adding additional substances to adjust the pH of the sample.

[0050] Lactoferrin can be extracted from any natural lactoferrin-containing material.Typically, the natural lactoferrin-containing material is milk, which is in abundant supply and is not contaminated with any processing aids (such as in whey from cheese making process).Fat-containing materials can adversely affect the lactoferrin binding capacity of the ion exchange process, so skim milk is preferred.Fat level should ideally be kept below 0.1%.

[0051] In embodiments where the natural lactoferrin-containing material is skim milk, the skim milk may be produced using a low-temperature bowl fat separation process, such a process avoiding the high temperatures of high-temperature bowl fat separation. Although such temperatures are only about 50°C (significantly lower than pasteurization temperatures), any heating of the lactoferrin while remaining in the milk matrix may best be avoided so as not to damage even a portion of the lactoferrin protein. Furthermore, bacterial growth is lower at low temperatures, which provides improved milk quality versus high-temperature bowl separation.

[0052] In the present invention, lactoferrin is extracted from natural lactoferrin-containing material.Any suitable extraction technique can be used for this step, provided that it is compatible with the results of the present invention.One suitable technique is to use cation exchange chromatography to extract lactoferrin from natural lactoferrin-containing material.

[0053] Generally speaking, ion exchange chromatography involves passing a native lactoferrin-containing material (e.g., skim milk) through a column. Lactoferrin binds to and is retained on the column while the remaining components of the milk matrix pass through the column. The retained lactoferrin can then be eluted using a different solvent, such as a sodium chloride eluent. In certain embodiments, such a process may utilize SP Sepharose Big Bead resin, which is composed of large cross-linked agarose beads (100-300 μm) modified with sulfonate (SP) strong cation exchange groups. During this process, the positively charged lactoferrin binds to the negatively charged resin.

[0054] In the present invention, the extracted lactoferrin is concentrated to produce a liquid concentrate with improved heat stability of lactoferrin.Similarly, any suitable technique can be used for this step, provided that it is compatible with the results of the present invention.One suitable technique for concentrating lactoferrin is ultrafiltration.

[0055] Generally speaking, ultrafiltration involves concentrating lactoferrin in a selected liquid carrier (e.g., RO water) while removing other contaminants (e.g., eluate from an extraction step). In certain embodiments, ultrafiltration membrane media designed to retain proteins with specific molecular weights (greater than 10-50 kDa) are used to concentrate lactoferrin.

[0056] The amount of lactoferrin in the liquid concentrate before pasteurization can be, for example, about 9-12 wt% (e.g., about 11% solids). This amount of lactoferrin is higher than that often present in conventional processes, which can range from 6-8 wt% lactoferrin. Advantageously, reconcentrating as it is done in the process of the present invention results in a higher percentage of lactoferrin in the concentrate, and the inventors have found that this allows the concentrate to be dried (e.g., by spray drying) to produce a powder with better particle size flexibility without the addition of a further concentration process.

[0057] In the present invention, the liquid concentrate is pasteurized by heating to a temperature effective for pasteurization for a time effective for pasteurization.Pasteurization is a well-practiced technique, and suitable pasteurization conditions for a given sample can be easily ascertained by those skilled in the art.Effective pasteurization can be achieved by heating the lactoferrin-containing liquid concentrate to a minimum temperature of 72°C for a minimum time of 15 seconds.Other forms of pasteurization are known in the art, and it is expected that the beneficial effects of the present invention can also be applied to such.

[0058] Finally, pasteurized liquid concentrate is dried under non-denaturing conditions to produce lactoferrin powder, in which substantially all of the lactoferrin is native lactoferrin.Traditional drying techniques include freeze-drying and spray-drying, and spray-drying is preferred in the present invention because it is generally better for producing particles with a relatively uniform particle size and does not usually require grinding the product, as is often the case with freeze-drying.Freeze-dried powders can also sometimes lack functionality, for example, they have a coarser particle size and may not be easily blended into milk powder.

[0059] Any suitable spray drying technique and equipment can be used. Typically, spray drying equipment allows for the manipulation of parameters including nozzle size, inlet and outlet temperatures, and residence time, all of which can be adjusted to produce powders with desired physical properties. We note that the physical properties of the resulting powder also directly affect its functional properties, such as its mixability and flowability with aqueous liquids.

[0060] Multi-stage spray drying may be advantageously used in the present invention because, among other benefits, it allows lower nozzle temperatures to be applied in the spraying and drying of the lactoferrin powder. As mentioned above, the shorter the time that the lactoferrin is exposed to relatively higher temperatures, the better.

[0061] For example, using a single stage dryer, the outlet temperature controls the outlet powder temperature. A multi-stage dryer, however, has the ability to manipulate the outlet powder temperature using secondary heating / cooling (secondary temperature manipulation is accomplished via a fluidized bed - the fluidized bed has its own heating and cooling source). A secondary drying system allows for less high drying temperatures at the outlet because the secondary drying system can complement and complete the drying at a lower temperature, thereby preserving the structure of lactoferrin. The inventors note that such spray drying may also contribute to improved health effects / functional benefits of lactoferrin in the powder, and that the cumulative effect of all of the steps in the process of the present invention may lead to a powder with improved functional lactoferrin.

[0062] It is within the ability of a person skilled in the art to adjust the various parameters of the spray dryer in order to produce a powder that does not denature lactoferrin and has the desired functional properties. Specific examples of two tests carried out by the inventors in this regard are described below.

[0063] Nozzle Size Test The inventors have found that the combination of various nozzle sizes, targeted nozzle pressures, and the use of a multi-stage spray dryer allows for the production of lactoferrin powders with specific and unique particle size distributions.

[0064] As described below, two exemplary particle size distributions are targeted, each of which is associated with a particular functional property in the finished product, i.e., mixability (powder and / or aqueous) and powder flowability. Specific elements of the spray dryer operation vary and include: (1) spray nozzle swirl chamber and orifice size combination, (2) main chamber inlet and outlet temperatures, (3) nozzle pressure, (4) degree of agglomeration, which is dictated in part by the re-entry point of the fines in the process, and (5) concentration of solids in the feed stream.

[0065] During these nozzle size tests, powder performance tests were performed in real time and parameters were adjusted in real time to achieve advantageous particle size distributions (i.e., finer and coarser variants as described below), allowing modifications to run conditions during testing and therefore immediate validation of the process.

[0066] The conclusion of the test resulted in two main variants, a finer powder and a coarser variant. The finer powder has a D of about 35-40 μm. 50 value and d of about 50 to 55 μm 90 The coarser variants are characterized by a D value of about 50-60 μm. 50 value and D of 125 to 130 μm 90 The required target aqueous miscibility and powder flow properties were achieved in the powder.

[0067] [Table 1]

[0068] Multi-Stage Spray Dryer The use of a multi-stage spray dryer (MSD) can greatly aid in preserving the native structure of lactoferrin powder by removing water via heated air through three stages: (1) a main drying chamber, (2) a static fluidized bed, and (3) a vibrating fluidized bed. In contrast to a single stage dryer, the MSD allows for further water removal after stage 1 via stage 2 and 3 processes. These processes allow for further removal of water to occur at lower temperatures, when the water activity has already been reduced and lactoferrin is susceptible to damage from higher temperatures. The static fluidized bed (stage 2) temperature never exceeds 75°C.

[0069] In contrast to MSD, it is not possible to match the temperature profile of drying when using a single stage dryer. A single stage dryer requires the use of either a higher drying temperature or a longer residence time to achieve similar results. This results in increased exposure to heat and increased damage to the native structure of lactoferrin powder when using a single stage dryer.

[0070] Percentage of solids in lactoferrin concentrate test A feed stream to the spray dryer with a lower solids content was also tested. An upstream ultrafiltration plant concentrated the lactoferrin concentrate to 10-12% solids before pasteurizing it and feeding it to the spray dryer.

[0071] A modified heating profile and spray pattern was applied to the feed stream, which allowed the mixability and flowability as well as particle size distribution defined above to be maintained while improving lactoferrin quantification. Typically, lowering the feed stream solids percentage affects the powder particle size distribution, decreasing the average particle size and downsizing the particle size distribution. Manipulation of other process parameters, including (1) nozzle swirl chamber, (2) nozzle orifice size, and (3) nozzle pressure, to maintain particle size distribution when spray drying lower solids percentage feed streams was found to result in increased lactoferrin recovery.

[0072] Pasteurization and spray drying were completed at the same concentration of solids, i.e., 10-12%. In contrast, prior art processes of which the inventors are aware typically pasteurize at lower solids (7-8%), apply evaporative concentration techniques, and then spray dry at a higher solids percentage. This allows a higher recovery percentage to be achieved, in line with teachings in the published literature, but with the above-mentioned detrimental consequences on the structural and biological functionality of the recovered lactoferrin. Thus, while other processes may achieve greater lactoferrin recovery than the process of the present invention by pasteurizing at a lower solids concentration, the present invention can allow the same recovery percentage by using a higher solids percentage before drying the concentrate.

[0073] A particular embodiment of the process of the present invention 10 will now be described in detail with reference to the flow chart shown in FIG. 1) Milk delivery / handling – to facility Whole milk is collected from the farm using double skinned road tankers. At pick-up, milk temperature is verified to ensure it meets regulatory requirements. Milk is delivered to the facility, unloaded and stored in refrigerated silos to a temperature of <5°C. Milk is monitored for deterioration to ensure it is processed within 24-48 hours of receipt at the facility. Milk temperature is monitored and milk quality is controlled via testing of milk for acidity levels with each tanker load delivered. 2) Fat Separation (Low Temperature Bowl Skimming) 12 Milk separation is a key process in milk processing plants. In this step, whole milk is separated into cream and skim fractions for further processing. Traditional separation involves the use of high temperature bowl sealed separators, which require the milk to be heated to 50°C during the separation process, which promotes bacterial growth. However, in the process of the present invention, a low temperature bowl milk separation technique is preferred, which allows for effective separation of cream from the skim fraction at a temperature of 24-26°C, ensuring that milk quality is maintained and minimizing bacterial growth during processing.

[0074] 3) Skim Pretreatment (Clarification / Filtration) Skim milk with a fat content of <0.1% is transferred to the lactoferrin process where it undergoes clarification and filtration. Clarification is performed utilizing the GEA Bacterial Removal System (BRS), a process designed to reduce the bacterial load of skim milk during processing. Bacteria of concern include spore formers such as Bacillus cereus, which is heat and cold tolerant up to 128°C, which can adversely affect the shelf life of milk for upstream processes. The milk is also filtered to ensure that excess fat and other impurities are removed prior to the ion exchange (i.e., lactoferrin removal) process in columns.

[0075] 4) Ion exchange process 14 Extraction of lactoferrin from skim milk is performed by an ion exchange process in which skim milk is passed through a column (holding vessel) containing a specific SP Sepharose Big Bead resin. The resin is composed of large cross-linked agarose beads (100-300 μm) modified with sulfonate (SP) cation exchange groups. It is designed for industrial applications, and the large particle size and physical stability of the base matrix allow the resin to be utilized in high volume commercial extraction processes. The ion exchange capacity of the resin binds the lactoferrin from the skim milk for collection through the elution process. The ion exchange process is a batch process that requires lactoferrin to be loaded onto the column until the loading efficiency falls below 93%. At this stage, a co-elution process is used to extract lactoferrin.

[0076] 5) Collection and storage of eluate Once the column loading process is complete, the lactoferrin is washed from the resin using a specially prepared buffer solution, which is a two-step elution process. The first part of the elution process uses a lower buffer concentration designed to remove undesirable protein impurities, consisting mainly of lactoperoxidase, but other impurities such as whey proteins and minerals are also removed at this point. Step two of the elution process uses a higher buffer strength to remove the lactoferrin from the Sepharose Big Beads and collect it in a storage container for further processing. The buffer conductivity level is controlled to ensure that impurities are removed, which will affect the final potency level (i.e., content and purity) of the lactoferrin product.

[0077] 6) Ultrafiltration 16 The accumulation of multiple elutions is a prerequisite for the ultrafiltration process. This step in the process is specifically designed to remove buffer reagents from lactoferrin until the desired conductivity level is reached in the lactoferrin solution. It is designed to remove impurities such as sodium, chloride, and nitrate. The filtration process then continues to achieve the final concentration of the product for pasteurization. The total solids and pH of the concentrate at the completion of this step are carefully controlled to maintain the native lactoferrin structure. The total solids and pH can be adjusted using process water, and other pH adjustment additives can be used at this point to ensure that the concentrate is within the required tolerable level.

[0078] 7) Pasteurization18 Pasteurization is specifically designed to ensure that food safety requirements are achieved in accordance with Australian regulations (Food Standards Australia and New Zealand). As described above, the pasteurization step is a critical step in maintaining the native level of lactoferrin protein and is designed to minimize the deleterious effects of temperature exposure on the lactoferrin concentrate. The concentrate must be heated to 72°C for a minimum of 15 seconds, and the process can be operated to ensure that minimum regulatory requirements are achieved while maintaining a minimum temperature difference between the heating medium and the product temperature. Cooling the lactoferrin concentrate immediately after this step can help maintain the bioactivity level of lactoferrin.

[0079] 8) Concentrate storage After pasteurization, the concentrate is stored below 5° C. and sufficient volume is accumulated to be processed through a fluid bed spray dryer. Continuous monitoring of the concentrate is performed during this process to ensure that total solids and pH are maintained within the tight limits necessary to maximize native lactoferrin production.

[0080] 9) Spray drying 20 The pasteurized lactoferrin concentrate is processed through a multi-stage spray drying process that gently dries the lactoferrin at a lower temperature than traditional spray drying processes. The first stage of the drying process is designed to reduce most of the moisture from the concentrate forming powder particles. This part of the drying process is specifically controlled using airflow, air temperature, and concentrate nozzle pressure to achieve powder particles specifically designed to meet customer needs and specifications. The second stage of drying consists of a drying fluidized bed that gently conditions the lactoferrin powder using lower temperature air and air flow rate to cool the protein matrix while gently adjusting the moisture content to the desired level. The product is then passed through a vibrating screen to ensure removal of larger particles / foreign objects before packaging.

[0081] 10) Packaging The final step in the process involves dispensing the lactoferrin powder into specially designed heavy foil pouches. The packaging material is carefully selected to ensure it has excellent barrier properties to ensure maintenance of lactoferrin product integrity for the extended shelf life of the product. The packaging format is typically 5 kg. However, this can vary depending on customer requirements.

[0082] The present invention also provides lactoferrin powder produced by the process described above, and food products comprising the lactoferrin powder produced by the process. Uses of these products include: Infant, follow-on and toddler nutrition Listed, evaluated and registered therapeutic and medicinal products (e.g. capsules, tablets, nasal sprays) Dietary supplements Adult Nutrition Sports nutrition Dairy products (drinks, yogurt, and powders) ●Functional food Nutritional and nutraceutical The physical characteristics of the lactoferrin powder produced by the process described above include the following: Smaller, more uniform lactoferrin particles allow for improved blending and solubility results Spray dried lactoferrin is lighter, fluffier particles, and more uniform in shape with low light scattering effect Liquidity can vary depending on customer needs and desired size Neutral / light taste and non-solidifying Sensitive to heat The color of spray-dried lactoferrin (i.e., light pink) lends itself better to dry blending versus freeze-dried lactoferrin because the color "blends" better with the base powder. As described above, the inventors have also discovered that the highly functional and biologically active lactoferrin powder described above has therapeutic activity. The experiments described below demonstrate that powdered lactoferrin produced according to the present invention has an inhibitory effect on viral replication and can stimulate an immune response in vivo, and the inventors believe that the results of these experiments allow for reasonable prediction of the therapeutic applications disclosed herein. Indeed, lactoferrin has been indicated as a potential therapeutic agent for use in the treatment or prevention of viral infections, including COVID-19, a disease caused by the SARS-CoV-2 virus, and the inventors expect that, in view of the advantageous biological and functional properties described above, lactoferrin powder produced according to the present invention, when used, will improve the efficacy of such treatment. In fact, the present invention provides lactoferrin in powder form, which retains substantially all of the biological activity of natural lactoferrin, and which also has beneficial functional properties (e.g., mixing properties, solubility, etc.) for formulation in pharmaceutical applications.

[0083] Thus, the present invention provides a method for preventing or treating a viral infection in a patient. In one method, a formulation containing lactoferrin powder produced as described above can be administered to a patient (e.g., via the patient's airway). In another method, a formulation containing lactoferrin powder produced as described above can be administered intranasally to a patient.

[0084] In other embodiments and aspects, a formulation comprising a lactoferrin powder produced as described above may be administered orally (e.g., in a capsule) to a patient. Also provided are nasal sprays comprising lactoferrin powder produced by the process described above, as well as pharmaceutical compositions comprising lactoferrin powder produced by the process and a pharma- ceutically acceptable excipient.

[0085] The inventors envision that lactoferrin-based nasal sprays can be used up to four times a day as prophylactic or post-exposure prophylaxis against viral infections. Given that lactoferrin is a highly conserved natural protein that is normally present in mucosal secretions, bovine lactoferrin is unlikely to be immunogenic and should be well tolerated (except for people with dairy allergies). This uniquely positions lactoferrin as a first-in-class nasal spray-delivered mucosal pan-pathogen inhibitor based on a highly conserved naturally occurring protein. Many synthetic virus-blocking nasal sprays have been developed, but they do not have the pleiotropic immune-enhancing functions of lactoferrin.

[0086] One particular therapeutic use envisioned by the inventors is as a potential preventative and adjunctive treatment for diseases caused by COVID-19 and other viral strains such as those that cause the common cold (rhinovirus) and influenza. Thus, experiments were conducted to investigate whether lactoferrin produced according to the present invention could be effective as a therapeutic agent against COVID-19 by examining whether it improves responses to COVID-19 in human bronchial epithelial cell studies and in human clinical trials. These experiments are described below. EXAMPLES

[0087] Example 1 - Production of lactoferrin powder A process according to an embodiment of the present invention is now described for preparing batches of lactoferrin powders, hereinafter referred to as PnF20224 and PnF21329 (see Example 2).

[0088] Milk was sourced from the Goulburn Valley in Victoria, Australia. Raw whole milk was separated and skim milk was transferred to a lactoferrin plant for processing. The skim milk was maintained at refrigerated temperatures throughout storage before being processed through a Bacterial Removal Separator (Clarifier) ​​which reduces the bacterial load in the raw skim milk, and then through a series of filters which remove any remaining foreign matter, fat, fat-soluble compounds, and insoluble matter. These filters become increasingly fine as the milk is processed through each, culminating in a 1 μm filter.

[0089] Extraction of lactoferrin from skim milk was performed through an ion exchange process, where the skim milk was passed through a radial flow column containing a specialized ion exchange resin. The resin is composed of large cross-linked agarose beads (100-300 μm) that are custom made for industrial use, and the large particle size and physical stability of the base matrix allows the resin to be utilized in high volume commercial extraction processes. The ion exchange capacity of the resin binds the lactoferrin from the skim milk for collection through the elution process.

[0090] The bound lactoferrin is eluted from the resin using a series of increasingly concentrated sodium chloride buffer solutions, the highest of which has a concentration of about 10% w / v. The lactoferrin eluate is collected and stored for further processing.

[0091] Ultrafiltration and diafiltration of the lactoferrin eluate removes sodium chloride ions from the lactoferrin solution and increases the solids content in solution. The pasteurization is specifically designed to ensure food safety requirements are achieved in accordance with Australian regulations (Food Standards Australia and New Zealand). The pasteurization step is designed to minimize the thermal profile of the lactoferrin concentrate. To meet regulatory requirements, the concentrate must be heated to 72°C for a minimum of 15 seconds or equivalent. Cooling the lactoferrin concentrate immediately after the pasteurizer can help maintain the bioactivity level of lactoferrin.

[0092] The lactoferrin concentrate was then processed through a multi-stage spray drying process that gently dries the lactoferrin at a lower temperature than conventional spray drying processes. The first stage of the drying process is designed to remove most of the moisture from the concentrate forming powder particles. This part of the drying process is specifically controlled using airflow, air temperature, and concentrate nozzle pressure to achieve powder particles specifically designed to meet customer needs and specifications. The second stage of drying consists of a drying fluidized bed that gently conditions the lactoferrin powder using lower temperature air and air flow rate to cool the protein matrix while gently adjusting the moisture content to the desired level. The product is then passed through a vibrating screen before packing.

[0093] The final step in the process involves dispensing the lactoferrin into specially designed heavy foil pouches. The lactoferrin powder is passed through a metal detector into the packaging material, which has excellent barrier properties and maintains the PUREnFERRIN product integrity for the extended shelf life of the product. The packaging format is typically 5 kg, but this can vary depending on customer requirements.

[0094] The parameters of the equipment used in the above described process are given in Table 1.

[0095] [Table 2-1]

[0096] [Table 2-2]

[0097] Example 2 - Comparison of lactoferrin powder of the present invention with commercially available lactoferrin powder The iron saturation level, characterization, quantification, and quality of lactoferrin in powder form produced according to the present invention were measured and compared to that of other commercially available lactoferrin powders (see Table 2). Iron saturation level, characterization, and quantification using cation exchange chromatography, reversed-phase high performance liquid chromatography (RP-HPLC), and mass spectrometry (MS) of finished bovine lactoferrin powders produced according to the present invention and commercially available from different manufacturers are described.

[0098] 1. Materials and Methods 1.1. Chemicals All reagents and chemicals used were of HPLC or analytical grade, including acetonitrile (FSBA955-4), water (FSBW6-4) Optima® LC-MS grade, trifluoroacetic acid with a purity of 99% (FSBT / 3258 / PB05-100mL), and Tris-hydrochloride (#BP153-500) purchased from Fisher Chemicals, Australia. Sodium chloride (#71380-5kg) was purchased from Sigma-Aldrich, Australia. Sodium hydroxide (#1.06469.1000) was purchased from Merck Millipore, Australia. Bovine lactoferrin standard (98.10% lactoferrin from bovine milk; product number: 127-04122; lot number: CAG5602) was purchased from Novachem, Australia.

[0099] 1.2. Iron and iron saturation analysis This method is used to determine the percentage of iron saturation of lactoferrin by measuring absorbance at 465 nm. At 100% saturation, the absorbance at 465 nm of a 10 mg / mL lactoferrin solution = 0.48. Based on this fact, Fe 3+, the saturation of an unknown solution can be estimated. To obtain a value between 1.00 and 1.50, the solution must be diluted for the A280 measurement. The solution must be filtered through a 0.45 μm cellulose acetate filter before measurements at A465 and A280. Analyses were performed by the Bureau Veritas Asure Quality (BVAQ) laboratory, North Melbourne, Victoria, Australia, using methods GB 5009.268.2-2016 (iron) and LFST 01 06.03 (iron saturation).

[0100] [Table 3]

[0101] 1.3.Sample preparation 1.3.1. Construction of Lactoferrin Standards and Calibration Curves Using Fast Performance Liquid Chromatography (FPLC) and Ultra-High Performance Liquid Chromatography (UHPLC) Lactoferrin reference material for establishing standards was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) with product number #127-04122 and lot number #CAG5602. Stock solution was prepared by dissolving 1 gram of lactoferrin in a 120 ml container containing 100 ml of MQ to obtain a concentration of 10 mg / ml. Samples were gently dissolved in an Intelli mixer (ELMI RM-2L) at room temperature for 3.5 hours. Working lactoferrin calibration standards were prepared from the stock solution to obtain working standards of 0.25, 0.5, 1, 2, 3, 4, and 5 mg / ml.

[0102] For UHPLC calibration, a stock solution was prepared at a concentration of 10 mg / ml by dissolving 200 mg of lactoferrin in 20 ml of MQ. Working standards were prepared by dilution of the stock solution in MQ. The concentrations used for the calibration curve were 0.05, 0.075, 0.1, 0.5, 1, 1.5, 2, and 2.5 mg / ml.

[0103] A calibration curve for lactoferrin was constructed using the relative responses of each to its respective target. 1.3.2. Commercial Bovine Lactoferrin Powder Sample Preparation for Cation Exchange Chromatography Analysis Using FPLC Lactoferrin powder stock solutions (10 mg / ml) were prepared by accurately weighing 200 mg of powder in a 50 mL Falcon tube. MQ water was added to the tube to a final volume of 20 mL or 20 g. Each powder was prepared in triplicate. Samples were gently dissolved in an Intelli mixer (ELMI RM-2L) for 3.5 hours at room temperature and passed through a 0.45 μm filter. Stock samples were diluted to 2 mg / ml for quantification using cation exchange chromatography (Capto HiRes cation exchange chromatography column, Cytiva).

[0104] Commercial Lactoferrin Powder Sample Preparation for MS and Callaghan Innovation RP-HPLC Analysis Lactoferrin powder stock solutions (10 mg / ml) were prepared by accurately weighing 100 mg of powder into a 50 mL Falcon tube. MQ water was added to the tube to a final volume of 10 mL or 10 g. Each powder was prepared in triplicate. Samples were gently dissolved in an Intelli mixer (ELMI RM-2L) at room temperature for 3.5 hours and passed through a 0.45 μm filter.

[0105] For both mass spectrometry and Callaghan Innovation RP-HPLC analysis, stock samples were diluted to 1.5 mg / ml. 1.4. Cation exchange chromatography Cation exchange chromatography experiments were performed on an analytical pre-packed Capto HiRes S 5 / 50 column (#29275877, Cytiva Lifesciences) for high-resolution separation of native lactoferrin protein using an AKTA pure 25 M2 FPLC system (GE Healthcare, USA) at room temperature. The gradient was followed by the protocol provided by GE Healthcare. The column was regenerated after every run according to the manufacturer's instructions.

[0106] 1.5.LC / Q-TOF MS and MS / MS analyzed were performed using an Agilent Technologies UHPLC 1290 Infinity Quaternary LC system equipped with a diode array detector (DAD) and an Advanced Bio 6545XT LC / Q-TOF instrument. Callaghan RP-HPLC analysis was performed on an Aeris™ 3.6 mm WIDEPORE XB-C8 200 Å analytical LC column (Part-OOG-4481-EO, 250x4.6 mm), C8 Security Guard Ultra Holder (Part-AJO-9000), and Security Guard Ultra Cartridges (Part-AJO-8771) from Phenomenex. Gradient elution was performed with a mixture of two solvents according to Billakanti et al. 2019 (14). Solvent A consisted of 0.1% trifluoroacetic acid (TFA) in water and solvent B was 0.1% TFA in acetonitrile. Detection was performed at a wavelength of 280 nm. Injection volumes consisted of 20-μl. At least three independent analyses were performed for each sample, and representative results are shown. Agilent Mass Hunter Qualitative Analysis Software 10 was used for data analysis.

[0107] MS analysis by 20-μl injection into an Agilent Technologies 6545XT Advanced Bio LC / Q-TOF instrument with Dual AJS ESI in positive electrospray ionization mode for retrieval and identification of lactoferrin intact protein from the samples. Ionization source conditions were set as follows: capillary voltage, 4.5 kV; source temperature, drying gas temperature 250° C., drying gas flow 8 l / min, nebulizer gas pressure 200 KPa (2 bar). Collision gas was high purity nitrogen. All LC-MS measurements were performed in triplicate. Agilent MassHunter BioConfirm 10 software and Sequence Manager software were used for data analysis.

[0108] 2. Results and Discussion 2.1. Iron content and iron saturation level The results show that total iron concentration and iron saturation levels vary in lactoferrin powders (Table 3). The iron content in lactoferrin powders is as follows: 105 mg / kg (Leprino Nutrition), 109 mg / kg (PnF20224), 129 mg / kg (PnF21329), 129 mg / kg (Tatura), 147 mg / kg (Nepean River Dairy), and 180 mg / kg (FUJIFILM Wako Pure Chemical Corporation), with Nepean River Dairy iron content ranked the highest.

[0109] The percentage of iron saturation of six different lactoferrin powders was calculated based on the ratio of light absorption at 465 nm and 280 nm measured by UV-Vis spectroscopy according to the BVAQ laboratory. The iron saturation levels ranged from 16% to 23% (Table 3). Typically, lactoferrin in its native form is characterized by 15-20% iron saturation and has a salmon pink color. Noumi and Leprino lactoferrin powders all contained 16%, Tatura was 14%, Nepean River Dairy was 23% (the highest of all analyzed), and the lactoferrin reference material from FUJIFILM Wako corporation was 19%.

[0110] The iron saturation level affects the physicochemical properties of the finished lactoferrin powder, which affects the function and effectiveness of lactoferrin, since lactoferrin function depends on its correct three-dimensional structure. Iron binding promotes lactoferrin changes in its three-dimensional structure and increases its thermal stability, with natural lactoferrin exhibiting slightly higher thermal stability than other forms. Noumi lactoferrin powder meets the salmon pink color criteria with 16% iron saturation, which is called natural lactoferrin, and therefore may be expected to exhibit slightly higher thermal stability.

[0111] [Table 4]

[0112] 2.2. Quantitative determination of lactoferrin in lactoferrin powder by cation exchange chromatography A high-resolution prepacked Capto HiRes S 5 / 50 column was selected for lactoferrin quantification because it provides excellent peak resolution. The results show that lactoferrin was sufficiently separated and eluted for accurate quantification. Chromatograms were recorded at 280 nm.

[0113] To determine lactoferrin concentration, a calibration curve was generated as described in the methods section. The reference lactoferrin generated a good linear calibration curve for the selected concentration range (0.25-5 mg / ml) with an R2 value of 0.9997. Following this, bovine lactoferrin powders from four different manufacturers were analyzed for quantification of lactoferrin content and purity using optimized assay conditions by GE Healthcare on a Capto HiRes S 5 / 50 column (Table 4).

[0114] Protein purity was determined based on the main lactoferrin peak percentage of the integrated chromatogram. Different lactoferrin purity and quantification levels were found in the lactoferrin powder samples (Table 3). Protein purity was calculated to be 98.76% and 98.58% for PnF20224 and PnF21329, 98.36% for lactoferrin (Nepean River Dairy), and 94.84% for lactoferrin (Tatura), respectively. The lactoferrin recovery percentages were 82% for PnF20224, 81% for PnF21329, 62% for Nepean River Dairy, and 74% for Tatura, respectively. The difference in lactoferrin recovery may be due to the different purification processes used by different lactoferrin commercial manufacturers.

[0115] [Table 5]

[0116] 2.3. Molecular weight of lactoferrin in commercially available lactoferrin powder A Q-TOF mass spectrometer was used to determine the molecular weight of lactoferrin present in commercial lactoferrin powders. The molecular weight of intact lactoferrin was measured for PnF20224, PnF21329, Nepean River Dairy, and Tatura. The results showed that they all matched the theoretical molecular weight of lactoferrin: PnF20224 was 83,194 Da, PnF21329 was 82,853 Da, Nepean River Dairy was 83,373 Da, and Tatura was 83,984 Da. The mass spectrometric results are presented in Table 5.

[0117] Previous studies have shown that lactoferrin has been isolated in multiple molecular weight forms from bovine colostrum and mature milk, designated as lactoferrin-a (molecular weight approximately 84,000) and lactoferrin-b (molecular weight approximately 80,000). These multiple mass forms naturally occur in various milk samples, with the different forms being due, at least in part, to different degrees of glycosylation. Noumi lactoferrin samples (i.e., those of the present invention) are both in the 83,000 Da range, indicating that Noumi lactoferrin is homogeneous in terms of molecular weight forms in the samples. Both Nepean River Dairy (NRD) lactoferrin and Tatura lactoferrin contain two or more molecular weight lactoferrin forms (NRD: 83,373 Da to 85,595 Da and Tatura: 83,984 Da to 88009 Da), indicating a lower quality of lactoferrin.

[0118] [Table 6]

[0119] 2.4. Lactoferrin Quantification Using Callaghan Innovation RP-HPLC Assay A Callaghan Innovation RP-HPLC analytical method was developed for the simultaneous determination of lactoferrin purity and quantity in commercial lactoferrin products. For the current experiment, the calibration curve was calculated using a correlation coefficient (R 2 ) value of 1 and was linear over the range of 0.05-2.5 mg / ml. The lactoferrin content in the lactoferrin powders tested was determined and summarized in Table 6. Lactoferrin recovery was 81.20% for PnF20224, 83.00% for PnF21329, 71.37% for Nepean River Dairy, and 81.79% for Tatura. Investigation of contaminating proteins in lactoferrin powders was outside the scope of this study, but Tatura lactoferrin powder was investigated by Lonnerdal et al. in 2020, who identified a minor lactoferrin fragment of approximately 25,000 Da along with the major LF protein of approximately 80,000 Da, indicating that lactoferrin was fragmented during the production process.

[0120] [Table 7]

[0121] In the current study, iron content, iron saturation levels, protein purity, and quantification (cation exchange and RP-HPLC) were evaluated for lactoferrin powders from Noumi Limited and two other Australian lactoferrin manufacturers (Nepean River Dairy and Tatura). All lactoferrins showed considerable differences in iron saturation, purity, and quantification. Both cation exchange chromatography and RP-HPLC analysis lactoferrin quantification reveal that both analytical techniques have individual advantages in testing different physiochemical properties that specify lactoferrin quantification. FPLC, cation exchange chromatography, and RP-HPLC results show differences in lactoferrin recovery, with Noumi lactoferrin (i.e., according to the present invention) having a higher recovery. Combining cation exchange and RP-HPLC techniques provides a better understanding of bovine lactoferrin isolated powder: cation exchange analysis provides insight into heat-induced changes to lactoferrin under native buffer conditions, whereas RP-HPLC does not provide such information (14, 21). Both quantitative chromatographic analyses demonstrated that Noumi lactoferrin powders (PnF20224 and PnF21329) had high recovery and demonstrated that the lactoferrin protein was stable during manufacturing conditions with minimal loss of biological activity, making them suitable for clinical studies, dietary supplements, and nutritional products.

[0122] Example 3 - In vitro investigation of antiviral activity A multifaceted research approach, conducting human lung cell studies, followed by in vivo rodent studies, and human clinical trials, aims to provide evidence of the therapeutic effects described herein.Unless otherwise noted, references to "lactoferrin" in Examples 3 and 4 refer to lactoferrin in the form of a lactoferrin-containing powder produced according to the methods of the present invention.

[0123] Ethics statement, donor recruitment, and pBEC collection. Primary bronchial epithelial cells (pBECs), obtained from healthy non-smoking donors during bronchoscopy with written informed consent, were provided by PABWark (The University of Newcastle). All subjects underwent fiberoptic bronchoscopy following standard guidelines. pBECs were obtained using a single coated nylon cytology brush applied under direct vision. Approximately 4–8 brushings were made through second- to third-generation bronchi.

[0124] Air-liquid interface culture of pBECs and conditionally reprogrammed pBECs Culture and differentiation at the air-liquid interface were performed as previously described (Loo SL, Wark PAB, Esneau C, Nichol KS, Hsu AC, Bartlett NW. Human coronaviruses 229E and OC43 replicate and induce distinct antiviral responses in differentiated primary human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 2020;319:L926-L931). pBECs obtained from a single asthmatic donor were expanded and differentiated to confluence prior to culture at the air-liquid interface. For pBECs obtained from healthy patients, cells were conditionally reprogrammed by co-culture with irradiated fibroblasts following a previously published protocol (Martinovich KM, Iosifidis T, Buckley AG, Looi K, Ling KM, Sutanto EN, Kicic-Starcevich E, Garratt LW, Shaw NC, Montgomery S, Lannigan FJ, Knight DA, Kicic A, Stick SM. Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics. Sci Rep 2017;7:17971). After 25-30 days of differentiation, cultures were confirmed as ready by the presence of pseudostratified structure, ciliated epithelium, and mucus / mucus producing cells.

[0125] Virus stocks and propagation OC43 virus stock was obtained from ATCC (VR-1558). American Type Culture Collection (ATCC) indicates that OC43 was propagated on HCT-8 cells at numerous unknown passages. ATCC-supplied OC43 titer was 2.8 x 105 median tissue culture infectious dose per milliliter (TCID50 / mL), and when the ATCC stock was received, the virus was passaged three times in MRC-5 cells to generate a working stock. OC43 titer was 2 x 108 TCID50 / mL and was used for testing according to WHO guidelines. Viral titer was measured by TCID50 assay in MRC-5 using the Spearman-Karber method.

[0126] Lactoferrin treatment and infection Bovine lactoferrin (LF) powder (Noumi Limited, Australia) was diluted in PBS to a stock concentration of 10 mg / mL. Upon full differentiation, apical treatment with LF was performed by addition of 50 μL of 100 μg / mL (LF100) and 1000 μg / mL (LF1000) diluted in BEBM minimal medium (MM). Differentiated pBEC were infected with OC43 at an moi of 0.1. Viral inoculum was diluted in 200 μL of MM and the corresponding concentration of LF was maintained during the exposure to LF during binding.

[0127] Prior to infection, cells were washed once with PBS and pre-treated for 3 hours with 50 μL of the corresponding concentration of LF (or MM control) only on the apical surface. After 3 hours, the virus inoculum was added directly to the wells without removing the LF treatment (total infection volume 250 μL). After 2 hours of incubation at 35° C., the inoculum was removed. The cells were washed with 500 ul of PBS to remove unbound virus. Finally, 50 uL of LF treatment was added apically, corresponding to the start of the time course. Treatment was maintained for the duration of the time course and pBECs were harvested at 0, 24, 48, and 96 hours post-infection. Where applicable (FIG. 5), 50 uL of LF treatment (or MM control) was added to the apical surface at 48 hours post-infection to refresh the treatment.

[0128] Sample collection and analysis from ALI cultures At the time of harvest, apical lavage was collected by addition of 450 uL of PBS for 5 min (or by addition of 400 uL when LF treatment was refreshed at 48 h post-infection) to measure infectious virus release. Semi-membranes were collected in RLT buffer containing 1% 2-mercaptoethanol (2ME) (Qiagen) for molecular analysis and in RIPA buffer containing protease inhibitor cocktail (Roche) for protein analysis. All samples were stored at -80°C until further use.

[0129] Measure of shed infectious virus by TCID50 OC43 working stock titers and released infectious virus in the apical wash were measured using the TCID end-point dilution method. MRC-5 were seeded at a concentration of 1.105 cells / well in 10% FCS EMEM medium (Hyclone) flat-bottom 96-well plates. The next day, when the cells reached 40% confluence, the cells were infected with samples, which were serially diluted in 1% EMEM medium. One row was kept as a control. The plates were incubated at 35°C for 5 days and examined using a microscope to determine the presence or absence of CPE in each column. These results were used to determine infectious virus units using the Spearman-Karber method.

[0130] Measure of viral load by qPCR Total RNA was extracted using miRNAeasy mini kit (Qiagen). Purified RNA was quantified by spectrophotometry (Nanodrop). cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (ABI). Plasmids for OC43 nucleocapsid gene PCR detection were used to generate standards for qPCR. Viral load qPCR analysis was performed using taqman assays targeting the OC43 N gene (Geneworks) using an ABI 7500 Real Time PCR System. All targets were normalized using 18S (Applied Biosystems, Thermofisher Scientific). Table 7 shows the primers and probes for these two assays.

[0131] [Table 8]

[0132] statistical analysis All data were analyzed using GraphPad Prism version 8.2.1 software. qPCR data (Figure 3) were compared by two-way analysis of variance with Sidak's multiple comparison test. Log-transformed infectious virus data measured by TCID50 (Figures 4 and 5) were compared by two-way analysis of variance with Sidak's multiple comparison test. P values ​​<0.05 were considered statistically significant.

[0133] result Human differentiated primary human bronchial epithelial cell assay As can be seen in Figure 2, apical treatment with lactoferrin showed a reduction in OC43 viral load over a 96 hour time course in pBECs obtained from one donor with asthma differentiated at the air-liquid interface. Cells were infected with OC43 at an moi of 0.1 in the presence or absence of lactoferrin treatment (LF100: 100 μg / mL; LF1000: 1000 μg / mL) diluted in minimal medium (MM). OC43 viral load was measured using an OC43-specific assay targeting the N gene. Data was normalized to the 18s housekeeper gene using an 18s-specific assay. N=1 As shown by Figure 2, single treatment of differentiated primary human bronchial epithelial cells with 1000 μg / mL lactoferrin (produced according to the present invention) resulted in 100% inhibition up to 4 days post-infection. A 10-fold lower dose of 100 μg / mL was also effective, showing 100% blockade of viral replication after 2 days and approximately 90% reduction at 4 days post-infection.

[0134] As can be seen in Figure 3, apical treatment with 1000 μg / mL lactoferrin also showed a significant reduction in OC43 viral load at 96 hours post-infection. pBECs obtained from two healthy donors were differentiated at the air-liquid interface. Cells were infected with OC43 at an moi of 0.1 in the presence or absence of lactoferrin treatment diluted in minimal medium (MM). OC43 viral load was measured using an OC43-specific assay targeting the N gene. Data were normalized to the 18s housekeeper gene using an 18s-specific assay. Statistical analysis was performed using a two-way ANOVA with Sidak's multiple comparison test, error bars represent the mean + / - SD; N=8; p<0.05 (* on graphs) Subsequent experiments showed that infectious virus was shed at the apical surface of airway epithelial cultures (Figures 4 and 5). As can be seen in Figure 4, apical treatment with 1000 μg / mL lactoferrin showed a significant reduction in OC43-released infectious virus at 48 hours post-infection. pBECs obtained from two healthy donors were differentiated at the air-liquid interface. Released infectious virus was measured by TCID50 assay using Spearman-Karber calculations, and logarithmic transformation of the data was analyzed by two-way ANOVA with Sidak's multiple comparison test. Cells were infected with OC43 at an moi of 0.1 in the presence or absence of lactoferrin treatment diluted in minimal medium (MM).

[0135] As can be seen in Figure 5, apical treatment with 1000 μg / mL lactoferrin showed a significant reduction in OC43-released infectious virus at 48 hours post-infection. pBECs obtained from one healthy donor were differentiated at the air-liquid interface. Cells were infected with OC43 at an moi of 0.1 in the presence or absence of lactoferrin treatment diluted in minimal medium (MM). Released infectious virus was measured by TCID50 assay using Spearman-Karber calculations and logarithmic transformation of data was analyzed by two-way ANOVA with Sidak's multiple comparison test. Error bars represent mean + / - SD; N=3; p<0.05 (* on graph). These data showed that LF1000 was statistically lower than untreated at 96 hours (lactoferrin supplemented at 48 hours).

[0136] Lactoferrin treatment at the time of infection significantly suppressed infectious virus production by more than 2 logs at 48 hours. Although the virus started to catch up in treated cells by 96 hours (Figure 4), it was still statistically lower (LF1000) than in untreated cells (Figure 5) when lactoferrin was supplemented at 48 hours, without immune system help. In vivo, lactoferrin is expected to provide a window of protection over the first days of infection while stimulating immunity to provide ongoing antiviral support. In vivo mouse studies (described below) are expected to illustrate this.

[0137] Thus, lactoferrin produced according to the invention reduces infection by human coronavirus (CoV) OC43 in an in vitro model using differentiated primary human bronchial epithelial cells (data are confidential but available for discussion upon request). These positive results are so striking that in vitro and in vivo (i.e. rodent models) studies are planned to investigate the effect of COVID-19 causing virus inhibition with a novel therapeutic modality (lactoferrin applied topically in the nasal cavity of animals) for application as an antiviral drug (e.g. for COVID-19) and over-the-counter drug.

[0138] Example 4 - In vivo investigation of antiviral activity in rodents Many viruses (including β-coronaviruses such as OC43 and SARS-CoV-2) use proteoglycans as co-receptors to facilitate binding to cells and promote infection. By binding to these cell surface proteoglycans, native lactoferrin has been shown to impede viral infection. In vitro (cell line) studies have reported the antiviral activity of lactoferrin against a range of respiratory viruses, including SARS-CoV-2. The experiments described below were performed to determine whether the lactoferrin in the lactoferrin powder prepared according to the present invention retained the biological activity of native lactoferrin.

[0139] As described above, the antiviral properties of lactoferrin have been demonstrated in differentiated primary human bronchial epithelial cells infected with epidemic human coronaviruses (e.g., OC43 and 229E). Subsequent experiments were performed to obtain early proof of concept of efficacy against SARS-CoV2 in this physiologically informative in vitro model of human airway epithelium.

[0140] The experiments described below were carried out using procedures that are standard in the art and well known to those skilled in the art. Intranasal treatment efficacy against OC43 infection in mice Rationale Antivirals may play an important role in the containment of COVID-19, as well as in the treatment for many of the current and future respiratory viral diseases. The antimicrobial / antiviral activity of native lactoferrin is proposed to be mediated by binding to sugar structures (e.g., heparan, sialic acid-based molecules) on the surface of cells that are recognized and used by many viruses for cell binding during infection. For example, human respiratory viruses such as coronaviruses utilize sugar-based molecular structures for cell binding. SARS-CoV-2 binds to heparan sulfate. The related human betacoronavirus OC43 utilizes the glycan-based receptor 9-O-acetylated sialic acid.

[0141] These experiments focus on testing the efficacy of intranasally delivered lactoferrin powder produced in accordance with the present invention (e.g., as described in Example 1) in blocking OC43 infection using a proprietary murine coronavirus (OC43) infection model developed in-house to provide proof-of-concept evidence supporting the development of a lactoferrin formulation as an anti-respiratory virus nasal spray. As previously noted, unless otherwise noted, references in this example to "lactoferrin" are to lactoferrin in the form of a lactoferrin-containing powder produced in accordance with the methods of the present invention.

[0142] the purpose 1. To determine whether intranasal lactoferrin treatment reduces infection by human coronavirus (CoV) OC43 in mice.

[0143] 2. To analyze the antiviral effects of lactoferrin and immune transcriptome biomarkers Test Design Three separate in vivo experiments were performed to provide proof-of-concept evidence supporting the development of lactoferrin formulations as anti-respiratory virus nasal sprays. A summary of the experimental design for each study is available in Table 8 and Figure 6.

[0144] Study 1: Lactoferrin Proof of Concept Mice (8 / group) were treated intranasally with lactoferrin at the time of infection and daily after infection (100 μg / mL*15 μL intranasally, corresponding to a total dose of 1.5 μg per mouse based on in vitro studies) to model post-exposure prophylaxis. After initial lactoferrin treatment, mice were treated with 1 × 10 6 The course of OC43 upper respiratory tract infection was modeled according to a constructed protocol by infection with TCID50 / mL OC43 (or UV-inactivated virus control - UV-OC43). Experimental endpoints were assessed at 2 and 48 hours post-infection. Nasal turbinates and nasal washes were collected for assessment of OC43 viral load by qPCR and infectivity by viral median tissue culture infectious dose (TCID50), respectively.

[0145] Test 2: Replication Mice (8 / group) were treated intranasally with lactoferrin at the time of infection and daily after infection (100 μg / mL*15 μL intranasally, corresponding to a total dose of 1.5 μg per mouse) to model post-exposure prophylaxis. In this replicate experiment, lactoferrin treatments were made fresh each day of the time course. After the initial lactoferrin treatment, mice were treated with 1×10 6 Mice were infected with TCID50 OC43 / mL (or UV-inactivated virus control - UV-OC43) to model the course of OC43 upper respiratory tract infection according to the established protocol. Experimental endpoints were evaluated at 2 and 48 hours post-infection. Nasal turbinates were collected for evaluation of OC43 virus load and innate antiviral immune responses (interferons IFN-β and IFN-λ2 / 3) by qPCR. Nasal washes were collected for evaluation of infectivity by virus median tissue culture infectious dose (TCID50), respectively.

[0146] Study 3: Immune transcriptome biomarkers Mice (8 / group) were treated intranasally with lactoferrin at the time of infection and twice daily after infection (10mg / mL*15μL intranasally, corresponding to a total dose of 150ug per mouse) to model post-exposure prophylaxis. After initial lactoferrin treatment, mice were treated with 1×10 6 Mice were infected with TCID50 / mL OC43 (or UV-inactivated virus control - UV-OC43) to model the course of OC43 upper respiratory tract infection according to the established protocol. Experimental endpoints were evaluated at 2 and 48 hours post-infection. Nasal turbinates were collected for evaluation of OC43 viral load by qPCR. Bronchoalveolar lavage was collected for leukocyte enumeration. Additionally, immune transcriptome biomarker units in nasal tissues were analyzed by Nanostring using the nCounter Mouse Immunology v2 Expression Panel from Nanostring (Nanostring, Seattle, WA), which evaluates the expression of n>500 common mouse immunology-related genes.

[0147] [Table 9]

[0148] result Study 1: Intranasal lactoferrin treatment showed a trend toward reduced OC43 viral load and infectious virus Figure 7A shows the OC43 viral load in nasal turbinates in untreated (vehicle) mice versus mice treated intranasally with 1.5 μg lactoferrin over a 48-hour time course. At 2 hours after virus inoculation, the OC43 viral load was comparable between vehicle and lactoferrin-treated mice. By 48 hours of infection, a 2-log increase in OC43 viral load was observed, indicating viral replication in the upper respiratory tract. For lactoferrin-treated mice, there was a trend toward reduced viral load at 48 hours after infection, as analyzed using a two-way ANOVA statistical test and Sidak's multiple comparison test (p=0.3368).

[0149] Figure 7B shows the measured OC43 infectious virus in MRC-5 cells using TCID50 assay in nasal wash samples. At 2 hours after virus inoculation, no significant difference in infectious virus dose was observed between untreated (vehicle) and LF-treated samples, but there was a trend toward reduced infectious virus in the lactoferrin-treated group (p=0.6727). For the infectious virus TCID50 assay, OC43 live infectious virus at 48 hours after infection was below the limit of detection (LOD) of the TCID50 assay.

[0150] As can be seen in Figure 7, OC43 viral load and infectious virus tended to decrease after intranasal lactoferrin treatment. Mice were treated intranasally with 1.5 μg lactoferrin at the time of infection and daily thereafter. A) viral load and B) infectious OC43 levels between vehicle and lactoferrin-treated mice were assessed by qPCR and TCID50 assay, respectively. Results were analyzed by two-way ANOVA with Sidak's multiple comparison test. N=8, LOD=limit of detection (63.2 TCID50 / mL).

[0151] Study 2: Intranasal lactoferrin treatment showed a trend toward reduced OC43 viral load at 48 hours post-infection, which was associated with reduced IFN mRNA. Because initial pilot studies showed trends toward reductions in OC43 viral RNA and infectious virus, the experiment was repeated. In contrast to the initial experiments, in which lactoferrin treatments were performed from pre-frozen aliquots of a stock lactoferrin concentration, lactoferrin powder provided by Noumi Limited was freshly diluted on each day of treatment.

[0152] OC43 viral load and infectious virus tended to decrease after intranasal lactoferrin treatment. Mice were treated intranasally with 1.5 μg lactoferrin at the time of infection and daily thereafter. A) viral load and B) infectious OC43 levels between vehicle and lactoferrin-treated mice were assessed by qPCR and TCID50 assay, respectively. Results were analyzed by A) two-way ANOVA with Sidak's multiple comparison test and B) unpaired T-test, n=8.

[0153] Similar results were observed for OC43 viral load as in the first study. Figure 8A shows the OC43 viral load in nasal turbinates in untreated (vehicle) mice versus mice treated intranasally with 1.5 μg lactoferrin over a 48-hour time course. Two hours after virus inoculation, OC43 viral load was comparable between vehicle and LF-treated mice. A 2-log increase in OC43 viral load was observed 48 hours after virus inoculation, indicating viral replication in the upper respiratory tract. There was a trend toward reduced viral load in LF-treated mice 48 hours after infection; two-way ANOVA statistical test and Sidak's multiple comparison test (p=0.2929).

[0154] Based on the results from Study 1, live virus was measured only at 2 hours post-infection because OC43 titers were below the detection limit of the TCID50 assay by 48 hours. Figure 8B shows measured OC43 infectious virus in MRC-5 cells using the TCID50 assay in nasal wash samples at 2 hours post-virus inoculation. A trend toward reduced infectious virus was observed in the LF treatment group (p=0.3748).

[0155] The innate antiviral immune response in nasal tissues was then investigated using in-house IFN-β and IFN-λ2 / 3 qPCR assays. The results are presented in Figures 9A and 9B for IFN-β and IFN-λ2 / 3, respectively. In Figure 9, the magnitude of innate immune gene expression following OC43 infection followed the viral load. Mice were treated intranasally with 1.5 μg lactoferrin at the time of infection and daily thereafter. A) IFN-β and B) IFN-λ2 / 3 levels between vehicle and lactoferrin-treated mice were assessed by qPCR. Results were analyzed by two-way ANOVA with Sidak's multiple comparison test. N=8 For both IFN molecules, there was a trend towards a reduction in mRNA levels at 48 h post-infection that was in line with the reduction in viral load observed at 48 h (Figure 4A); analyzed using a two-way ANOVA statistical test and Sidak's multiple comparison test (p=0.25139 and 0.5183 for IFN-β and IFN-λ2 / 3, respectively).

[0156] Study 3: Intranasal lactoferrin treatment The final study investigated the antiviral efficacy of intranasal lactoferrin treatment at a 100-fold higher dose and more frequent administration. In this experiment, mice were treated intranasally with 150 μg lactoferrin and received a total of five intranasal doses by 48 hours post-infection. Two additional control groups were added in the experimental design: UV-OC43 / untreated (mock vehicle) and UV-OC43 / LF treated (mock+LF).

[0157] Viral RNA was below the detection limit in nasal turbinates of the UV-OC43-infected group, indicating that viral RNA levels in nasal tissues were due to live virus rather than persistent detection of the initial inoculum. In the OC43-infected group, we observed a 1 log increase in OC43 viral load between 2 and 48 hours of infection for both vehicle- and lactoferrin-treated groups. Finally, there was no difference in viral load between vehicle and LF-treated groups at both the 2 and 48 hour time points.

[0158] The lactoferrin dose in this study was 100 times higher than that in the previous study, in order to establish a higher level of resistance and efficacy. However, this appears to be too high, and no effect on viral load was observed. We note that, for example, it may be the case that the increased volume of nasal spray simply falls down the mouse's nasal passages. Future experiments will be only twice as high (or so).

[0159] Leukocyte recruitment in bronchoalveolar lavage (BAL) was then investigated to assess the ability of upper airway lactoferrin treatment to generate protective immune cell recruitment in the lower airway. As can be seen in Figure 10, OC43 infection and lactoferrin intranasal treatment. Mice were intranasally treated with 150 μg lactoferrin at the time of infection and twice daily thereafter, and BAL (A) macrophages, (B) neutrophils, and (C) lymphocytes were enumerated between vehicle and lactoferrin treatment groups, as well as between mock-infected and infected groups. Results were analyzed in a "mixed effects" statistical test using Tukey's multiple comparison test. n=8 for each group.

[0160] Figure 10A shows macrophages in the lower respiratory tract. At 2 hours post-infection, significant differences between groups were observed. Both groups inoculated with UV-inactivated OC43 showed significantly higher macrophage counts compared to the OC43-infected group (p<0.01). Additionally, the OC43 lactoferrin-treated group tended to have lower macrophage counts than its untreated counterpart; mixed-effects statistical test with Tukey's multiple comparisons (p=0.1337). By 48 hours post-infection, there were no differences in macrophage counts within all groups. Figure 10B shows BAL neutrophil counts at 2 and 48 hours post-OC43 inoculation. There was no significant difference in neutrophil counts for both time points. Figure 10C shows lymphocyte counts in the lower respiratory tract. After 2 hours, lymphocyte counts in the mock-infected untreated group were significantly lower than the OC43-infected vehicle group (p<0.05). There was a trend toward increased lymphocytes in the lactoferrin-treated group compared to the mock-infected and vehicle-treated groups (p=0.2006 and p=2101 for the OC43-vehicle and OC43-lactoferrin-treated groups, respectively). By 48 hours post-infection, there were no differences in lymphocyte counts.

[0161] These data indicate that more regular administration of lactoferrin may be required. In humans, nasal sprays four times a day are required for efficacy in preventing viral infection. Future experiments will treat mice more frequently, in line with what occurs in typical human treatment regimens.

[0162] Study 3 continued: Lactoferrin immune transcriptomic biomarkers To investigate lactoferrin immunomodulatory activity, nasal tissue immune transcriptome biomarker units were analyzed using the Nanostring Mouse Immunology v2 Expression Panel (Nanostring, Seattle, WA), which evaluates the expression of n>500 common mouse immunology-related genes. Uninfected and untreated groups were compared to uninfected lactoferrin groups, and two outliers (one in the vehicle-treated group and one in the lactoferrin-treated group) were removed for this analysis.

[0163] Nanostring revealed upregulation of biomarkers involved in T cell signaling and antigen presentation, lymphocyte recruitment (IL20 CXLC13 and apoptosis (caspase 8), immune system pathways from macrophages and T cells (CD2, CD22, ITG B2), leukocyte migration pathways (ITGB2, ICAM 2 ITGA4.CD2, SELL), and JAK-STAT signaling pathway (SHP1, STAT), as well as hematopoietic cell lineages (CD127, HLA-DR, CD19, CD22, CD20).

[0164] Data were further analyzed for pathway enrichment with lactoferrin treatment. Lactoferrin promoted enrichment of multiple pathways surrounding lymphocyte recruitment, binding, and maturation in antigen presentation and signaling pathways (TLR / NFkB / IFN / TNF signaling).

[0165] Finally, this analysis revealed that intranasal lactoferrin treatment promoted nasal immune cell recruitment. Compared to the vehicle-treated group, the LF-treated group was enriched for CD45 (a leukocyte marker), B cells, and low levels of neutrophils and T cells. The one exception was cytotoxic T cells.

[0166] conclusion In this report, the efficacy of intranasally delivered lactoferrin against OC43 infection was investigated using a murine coronavirus (OC43) infection model developed to provide proof-of-concept evidence supporting the development of a lactoferrin formulation as an antirespiratory virus nasal spray.

[0167] This was carried out across three in vivo studies to determine whether intranasal lactoferrin treatment reduced infection with human coronavirus (CoV) OC43 in mice and to perform an analysis of lactoferrin immune transcriptomic biomarkers.

[0168] Studies 1 and 2 focused on the antiviral effect of lactoferrin treatment by examining viral load and infectious virus between vehicle and 1.5 μg lactoferrin treated mice. Importantly, in both studies there was a strong trend toward reduced viral load at 48 hours post-infection.

[0169] In a third study, where the lactoferrin dose was increased 100-fold (from 1.5μg to 150μg), the viral loads between the vehicle-treated and OC43-treated groups were similar, indicating that the lactoferrin dose increased from 1.5μg to 150μg may be too high and failed to effectively prevent infection. Testing additional treatment doses, such as 15ug (i.e., only 10-fold higher), is the next step in determining the optimal dose for preventing OC43 viral load by intranasal lactoferrin treatment.

[0170] When looking at infectious OC43 in nasal wash samples (studies 1 and 2), OC43 binding at 2 hours post-inoculation showed a trend toward a reduction in lactoferrin-treated groups compared to vehicle-treated groups. Further testing with increased numbers of mice (in addition to treating earlier and more frequently with lactoferrin, similar to models of treatment in humans) would help provide the power necessary to reach statistical significance. At 48 hours post-infection, infectious viral units were below the detection limit of the assay. The lack of infectious OC43 by 48 hours post-infection, in contrast to the increase in viral RNA observed by qPCR, is likely an artifact of the experimental model, which needs to be further optimized.

[0171] Study 3 further focused on the evaluation of immune biomarkers after lactoferrin treatment at a higher dose (150 μg per treatment) and at a more frequent dose (twice a day), although twice a day may still be too little treatment, as in human nasal spray applications, and four times a day is typical in terms of frequency of application. The objective of Study 3 was to first determine whether upper airway lactoferrin treatment could generate protective immune cell recruitment in the lower airways by enumerating leukocytes in bronchoalveolar lavage. Two hours after infection with OC43 in the upper airways, a significant recruitment of lymphocytes, but not macrophages or neutrophils, was induced (p<0.05). At 48 hours after viral challenge, there was no change in white blood cell counts in the OC43 vehicle-treated and OC43 lactoferrin-treated groups compared to UV-OC43 controls, which may indicate that changes in white blood cell counts may be related to only a transient perturbation or that such effects may be related to early immune system "priming."

[0172] Finally, nasal tissue immune transcriptome biomarker units were analyzed between vehicle- and LF-treated groups using the Nanostring Mouse Immunology v2 Expression Panel (Nanostring, Seattle, WA). Lactoferrin treatment promoted MHC-II expression, T and B cell markers, and CXCL13 production, a cytokine that plays a key role in B and T cell homing. This result indicates that these may be suitable biomarkers in nasal brushing. Pathway enrichment analysis revealed that lactoferrin intranasal treatment promoted enrichment of multiple pathways surrounding lymphocyte recruitment, binding, and maturation, with antigen presentation and signaling pathways (TLR / NFkB / IFN / TNF signaling). This was further complemented by enrichment cores for immune cell populations such as B and T cells, neutrophils, and enriched CD45 (leukocyte marker).

[0173] method OC43 growth, purification, and quantification The original OC43 (ATCC number VR-1558) passage history is unknown and is propagated on HCT-8 cells. The initial stock from ATCC was received at a concentration of 2.8 x 105 TCID50 / ml and passaged three times in MRC-5 cells to generate a working stock as previously described (7). The MRC-5 cell line was used for TCID50 assays using the Spearman-Karber method to quantify viral load.

[0174] Virus challenge and treatment administration Six- to eight-week-old female BALB / c mice were obtained from Australian Bioresources (Moss Vale, Sydney, NSW). Treatment and virus challenge were performed under light anesthesia using isoflurane. Mice were challenged intranasally with OC43 at a concentration of 1.15×106 TCID50 in 15 μL (studies 1 and 2) or 10 μL PBS to model upper respiratory tract infection. UV-OC43 used in study 3 was inactivated as previously described for respiratory viruses.

[0175] Nasal wash, bronchoalveolar lavage, and cell analysis Mice tracheas were cannulated, the upper airways were irrigated with HBSS (HyClone, GE Life Sciences), and nasal washes were collected at the nostrils. Nasal washes were stored at -80°C. Bronchoalveolar lavage was processed for leukocyte and cytokine enumeration as previously described.

[0176] RNA extraction and reverse transcription Nasal turbinates were excised and vortexed for 30 seconds in RLT containing 1% beta-mercaptoethanol. Turbinate debris was removed and lysates were stored at -80°C. RNA was extracted using the miRNAeasy kit (Qiagen) according to the supplier's protocol. RNA was measured by spectrophotometry (Nanodrop) and reverse transcription reactions were performed with the High-Capacity cDNA Reverse Transcription Kit (ABI) using 500 ng of RNA according to the manufacturer's recommendations.

[0177] quantitative PCR Quantitative PCR (qPCR) was performed on a QuantStudio 6 using TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) and primer-probe combinations (Thermo Fisher Scientific) as outlined (Table 9). Standards of known concentration were used for absolute quantification of genes of interest. 18s was used as a reference gene to normalize the copy number of genes of interest.

[0178] [Table 10]

[0179] Viral median tissue culture infectious dose (TCID50) viral infectivity assay TCID50 assays for OC43 coronavirus were performed in MRC-5 cells using 10% FCS EMEM growth medium and 1% FCS EMEM assay medium. MRC-5 cells were resuspended in growth medium and seeded in 96-well plates (1 × 105 cells per 100 μL per well). At 30-40% confluence (usually after overnight incubation), cells were infected with nasal wash samples, starting with 2-fold dilutions followed by 10-fold dilution steps in EMEM containing 1% FCS. After 5 days of incubation at 33 °C / 5% CO2, plates were examined for cytopathological effects by light microscopy. The virus titer of each sample was then determined using the Spearman-Karber method.

[0180] Immune transcriptome expression analysis Nasal turbinate RNA was hybridized to nCounter Mouse Immunology Panel (NanoString) and processed on a Nanostring Prep-station according to the manufacturer's instructions at high sensitivity binding settings. 555 fields were counted on an nCounter Digital Analyzer. Raw data was quality control (QC) checked in nSolver Analysis software 4.0 for content normalization QC and normalized to positive / negative controls and housekeeper gene expression. Raw counts were imported into nSolver Advanced Analysis software (v2.0.134) for automatic normalization using GENorm software and pathway / cell enrichment analysis. The advanced analysis platform identified DEGs using linear regression models. DEGs were volcano plotted in GraphPad Prism 9.1.2. Heatmapper software was used to generate visual representations of the average Euclidean clustering of samples and gene expression patterns, as well as pathway enrichment z-scores and cell enrichment profiles based on the Nanostring advanced analysis output.

[0181] It will be apparent to those skilled in the art of the present invention that many modifications can be made without departing from the spirit and scope of the invention. In the following claims and the preceding description of the invention, unless the context otherwise requires by express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in their inclusive sense, i.e., to specify the presence of described features in various embodiments of the invention but not to exclude the presence or addition of further features.

Claims

1. 1. A method for producing lactoferrin powder, comprising: Extracting lactoferrin from a natural lactoferrin-containing material and concentrating the extracted lactoferrin to produce a liquid concentrate in which the extracted lactoferrin has improved stability, the liquid concentrate having a pH of 5.2 to 7.2 and a concentration of lactoferrin of about 9 to 12 wt %; heating the liquid concentrate to a temperature and for a time effective to pasteurize the liquid concentrate; drying the pasteurized liquid concentrate under non-denaturing conditions to produce a lactoferrin powder, wherein substantially all of said lactoferrin is native lactoferrin.

2. 10. The method of claim 1, wherein the extracted lactoferrin has improved stability due to the absence in the liquid concentrate of species that destabilize the lactoferrin.

3. 10. The method of claim 1, wherein the extracted lactoferrin has improved stability due to the presence in the liquid concentrate of species that stabilize the lactoferrin.

4. The method of claim 1 , wherein the liquid concentrate comprises one or more of a pH adjuster, a stabilizing mineral, and a dissolved gas.

5. The method of claim 1 , wherein the natural lactoferrin-containing material is milk.

6. 2. The method of claim 1, wherein the natural lactoferrin-containing material is skim milk.

7. 7. The method of claim 6, wherein the skim milk is produced using a cold bowl fat separation process.

8. 10. The method of claim 1, wherein the lactoferrin is extracted from the natural lactoferrin-containing material using ion exchange chromatography.

9. 9. The method of claim 8, wherein a two-step elution is performed on the ion exchange chromatography, the first elution step comprising eluting the species immobilized on the ion exchange column using a buffer solution having a relatively low concentration, and the second elution step comprising eluting the lactoferrin immobilized on the ion exchange column using a buffer solution having a relatively high concentration.

10. 10. The method of claim 1, wherein the extracted lactoferrin is concentrated using ultrafiltration.

11. 10. The method of claim 1, wherein the pasteurized liquid concentrate is cooled immediately after pasteurization.

12. 10. The method of claim 1, wherein the pasteurized liquid concentrate is dried by spray drying.

13. 13. The method of claim 12, wherein the spray drying comprises multi-stage spray drying.

14. 10. The method of claim 1, wherein greater than 90% of the lactoferrin in the lactoferrin powder is native lactoferrin.

15. 10. The method of claim 1, wherein the lactoferrin powder consists essentially of lactoferrin.

16. 10. The method of claim 1, wherein the lactoferrin powder has a D50 particle size of about 50 μm.

17. A lactoferrin powder produced by the method of any one of claims 1 to 16.

18. A food product comprising lactoferrin powder produced by the method of any one of claims 1 to 16.

19. A nasal spray comprising lactoferrin powder produced by the method of any one of claims 1 to 16.

20. A pharmaceutical composition comprising lactoferrin powder produced by the method of any one of claims 1 to 16 and a pharmaceutically acceptable excipient.

21. 21. The pharmaceutical composition of claim 20 for preventing or treating a viral infection in a patient.

22. The pharmaceutical composition of claim 21, wherein the prevention or treatment of the viral infection comprises administering the pharmaceutical composition via the patient's respiratory tract.

23. 21. The pharmaceutical composition of claim 20 for nasal administration to prevent or treat a viral infection in a patient.

24. The pharmaceutical composition of claim 21 for preventing the viral infection.

Images