GB2601226A

GB2601226A - Method for the preparation of water soluble eggshell membrane derived peptides

Info

Publication number: GB2601226A
Application number: GB2114135.3A
Authority: GB
Inventors: Barham Paul; Schmidt Ralf; Suso Henri-Pierre
Original assignee: Biovotec As
Current assignee: Biovotec As
Priority date: 2020-10-02
Filing date: 2021-10-01
Publication date: 2022-05-25
Also published as: GB202015658D0; GB202114135D0

Abstract

A method for preparing water soluble peptides from eggshell membrane (ESM) comprising providing an at least 20% w/v mixture of eggshell membrane and an aqueous solution of a strong base (e.g. sodium hydroxide) having a pH of at least 13; incubating the mixture at a temperature of about 110oC to 130oC and at a pressure of at least 150kPa for about 10 to 30 minutes; separating insoluble residues from the peptide solution and neutralising the solution. The neutralised solution may be diluted or filtered to remove cellular organisms. Further provided are compositions comprising the water soluble eggshell membrane peptides obtained from the method and the use of the same in therapeutic, nutritional and cosmetic applications. The water soluble eggshell membrane peptides obtained from the method may also be used in in vitro biological contexts, e.g. cell culture.

Description

Method for the preparation of water soluble eggshell membrane derived peptides The present invention relates generally to water soluble eggshell membrane derived peptides, methods for their preparation and uses thereof. More specifically, the present invention relates to a simple method for preparing water soluble peptides from eggshell membrane based on the rapid alkaline hydrolysis of water insoluble eggshell membrane under elevated pressure and few subsequent purification and neutralisation steps. The process is time-efficient, resource-efficient, high-yielding, economical and amenable to scale-up. The invention further provides compositions comprising the water soluble eggshell membrane peptides obtained from the method and the use of the same in therapeutic, nutritional and cosmetic applications. Beyond uses in or on the human or animal body the water soluble eggshell membrane peptides obtained from the method may be used in in vitro biological contexts, e.g. in cell, tissue and organ culture systems.

Humans and animals have used eggs as a food source for millennia. Avian eggs, for example chicken eggs, provide a vital assortment of essential nutrients. These eggs consist of a series of components: the egg shell, the vitellus (yolk), the albumin (egg white), as well as a variety of thin membranes. All of these components provide a source of valuable biomaterials including calcified compounds, proteins (including collagen), lipids and carbohydrates that have widespread applications in medical, health and cosmetic industries. While these key biomaterials can be readily isolated from the egg shell, yolk and albumin components of the egg, there remains a difficulty in isolating the valuable source of biomaterials found in the egg membranes, particularly the eggshell membrane. As a result, the additional biomaterial found in the eggshell membrane is often considered a waste product, but in principle non-reduced and bioactive water soluble peptides derived from eggshell membrane would find utility in a wide-range of industrial applications including therapeutic, nutritional and cosmetic contexts and also in vitro biological contexts, e.g. in cell, tissue and organ culture systems.

A major drawback in the processing and isolation of biomaterials (e.g. proteins) from the eggshell membrane is the difficulties faced in solubilising the starting -2 -material in a manner that balances high yields of non-reduced, bioactive components with cost-effectiveness and time-efficiency.

Previous approaches have included the use of acid hydrolysis, hydrolytic enzymes, mercaptopropionic acid or other harsh extraction agents. The acid hydrolysis of proteins is a well-established procedure, but when applied to eggshell membranes which contain proteins cross-linked by numerous disulphide bonds the acid treatment that breaks down the peptide bonds to liberate soluble peptides and amino acids also reduces the disulphide bridges present in the protein structures and generates a strong smell of hydrogen sulphide (rotten eggs). Acid hydrolysis is also known to be difficult to control and can break the protein down into its constituent amino acids thus losing the potentially valuable bioactivity of larger peptides. US 8197852 instead proposes an approach based on a prolonged mild alkaline hydrolysis step and numerous downstream physical and chemical processing steps including a step to concentrate the peptide solution. There are also methods and processes known in the art that involve the use of strong alkali to solubilise eggshell membrane (e.g. JP2018016610, CN103275205, CN102399281, JP2014040402, JPH11193279, JPH10158646 and JPH0940564). However, these approaches all have various problems associated with them, including time and cost inefficiencies and lack of scalability, isolation of small amounts of water soluble proteinaceous material and the inability to hydrolyse large amounts of insoluble starting material, unwanted sulphide reduction leading to unpleasant odour, loss of native activity, and/or the need to add chemicals to deal with by-products associated with unpleasant odours.

There is an ongoing need therefore for more time-efficient, cost-effective, resource-efficient, high-yielding and scalable methods of preparing water soluble eggshell membrane peptides which retain native structures and bioactivity so that the product so prepared can be utilised industrially. The present invention seeks to meet these needs by providing a method for preparing high yields of highly-enriched, non-reduced, bioacfive water soluble peptides from eggshell membrane based on a rapid step of strong base hydrolysis of high concentrations of water insoluble eggshell membrane under elevated pressure followed by a limited number of simple purification and neutralisation steps. -3 -

Thus, in a first aspect, the invention provides a method for preparing water soluble eggshell membrane (ESM) peptides, wherein said method comprises: (a) providing an at least 20% w/v mixture of water insoluble eggshell membrane and an aqueous solution of a strong base, wherein said mixture has a pH of at least 13.0 and is essentially free of eggshell (b) incubating said mixture at a temperature of about 110 °C to about 130°C and at a pressure of at least 150kPa for about 10 to about 30 minutes so as to form an aqueous solution of water soluble eggshell membrane peptides; (c) separating insoluble residues from the peptide solution formed in step (a); and (d) neutralising the residue-depleted peptide solution; and optionally (e) diluting the neutralised residue-depleted peptide solution with an aqueous liquid to a physiological conductivity, optionally wherein the neutralised residue-depleted peptide solution or the diluted neutralised residue-depleted peptide solution is filtered to remove cellular microorganisms.

As used herein, the term "peptide" includes any number of amino acids linked chemically via by peptide (amide) bonds. Thus, the term specifically includes dipeptides, tripeptides, tetrapeptides, pentapeptides, but also peptides which may be described as oligopeptides (e.g. those peptides of up to 25, 50, 75 or 100 residues) and polypeptides/proteins. The term further includes peptide conjugates in which two or more peptides are chemically linked by covalent bonds, e.g. disulphide bonds, to form multimers, e.g. dimers and timers. The term still further includes peptide aggregates in which two or more peptides are linked by non-covalent bonds, e.g. ionic bonds and van der Waals forces, to form multimers, e.g. dimers, trimers, tetramers and pentamers,. The peptide solution may also comprise individual amino acids.

The amino acids in the peptide solution, both free and as part of the peptides, will typically be selected from the naturally occurring amino acids on account of their natural origin, although minor modification from the alkaline hydrolysis process may -4 -occur. In certain embodiments, however, the presence of sulfhydryl (thiol) groups will be minimised. In preferred embodiments such groups will be substantially absent, or at least present in amounts no greater than in the eggshell membrane starting material. In most preferred embodiments such groups will be essentially absent, i.e. undetectable by routine means. Viewed alternatively, the number of disulphide bonds in the peptide solution will be essentially the same, or no less than that in the eggshell membrane starting material.

Water soluble peptides can be considered to be peptides for which less than about 1000 parts pure water are required to solubilise 1 part of the peptide at 25 °C and atmospheric pressure (101325 Pa) In other embodiments less than about 500, e.g. less than about 250, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 parts pure water are required to solubilise 1 part of peptide.

Expressed differently, the aqueous solution of water soluble eggshell membrane peptides formed in step (a) may have a peptide concentration of at least 5%, e.g. at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or at least 60% w/v.

"% w/v" (or "percentage weight by volume") is a commonly used expression of the amount of a certain compound or material in a liquid mixture, e.g. solution. 1% w/v equates to 1 gram of the compound or material per 100m1 of liquid, 2% w/v equates to 2g of the compound or material per 100m1of liquid, and so on. Accordingly, % w/v may be expressed as g/100m1, grams per 100 ml and g 100mI-1. 1% w/v also equates to 10 gram of the compound or material per litre of liquid. The skilled man would understand that through appropriate scaling calculations, % w/v can be expressed in terms of any SI unit of mass.

"% w/w" (or "percentage weight by weight") is a commonly used expression of the amount of a particular compound or material in a composition, typically a solid composition. 1% w/w equates to 1 gram of the compound or material per 100g of composition, 2% w/w equates to 2g of the compound or material per 100g of compound, and so on. Accordingly, % w/w may be expressed as g/100g, grams per 100g and g 100g-1. 1% w/w also equates to 10 gram of the compound or material per kilogram of solid. The skilled man would understand that through appropriate scaling calculations, % w/w can be expressed in terms of any SI unit of mass. -5 -

Eggshell membrane (ESM) is the fibrous bilayer found between the albumen and the eggshell of avian eggs, e.g. the eggs of fowl (gamefowl/landfowl (Galliformes) and waterfowl (Anseriformes)) and poultry, in particular, pigeon, pheasant, partridge, grouse or gull. The eggs of Gallus gal/us domesticus, the domestic chicken, are especially preferred. Either or both layers of the bilayer may be used in accordance with the invention.

The ESM of use in the invention will be in a form which is substantially, e.g. essentially, insoluble in water at a neutral pH, e.g. pH 6.8-7.2. For the purposes of the invention an insoluble material requires greater than 10L of solvent to dissolve 1g of solute. Such forms include sheets, flakes, shreds and powders of ESM. The reference to powdered ESM includes references to particulate ESM and ESM particles. In certain embodiments the ESM of use in the invention is also insoluble in organic solvents.

The insoluble ESM may be provided in dry or wet form. Flaked or shredded ESM in wetted form may be referred to as a "wet pulp" or simply a "pulp". If a wet form is used, the volume of any solution containing the strong base to be used should account for the water held up in the pulp to ensure an appropriate pH is obtained and that a resulting peptide solution of sufficient concentration is obtained. Likewise, if a wet form is used the optional downstream dilution step can be avoided if the wet form contains a sufficient amount of water.

The insoluble ESM to undergo alkaline hydrolysis in step (a) is essentially free from eggshell. Expressed numerically, the calcium carbonate content of the insoluble ESM should be no more than 8% w/w, e.g. no more than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% w/w.

Preferably, the insoluble ESM to be incubated with the strong base is essentially free of other (non-ESM and non-eggshell) egg components (which may be considered "contaminating" substances vis a vis the ESM), e.g. albumen and/or yolk. By "essentially free" it is meant that the ESM to be incubated with the strong base contains no more than 10% w/w, e.g. no more than 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% w/w of non-ESM and non-eggshell egg components.

In certain embodiments the ESM to be incubated with the strong base has a protein content of at least 80% w/w, e.g. at least 85%, 90%, 95% or at least 99% w/w. -6 -

Protein content may be determined by routine analysis including the Dumas method, the Kjeldahl method, and combustion analysis.

More specifically the ESM to be incubated with the strong base will be chemically substantially non-degraded, non-digested (e.g. chemically or enzymatically), non-reduced and/or non-denatured as compared to naturally occurring ESM from a corresponding avian source. By "substantially non-degraded" it is meant that less than 20%, e.g. less than 15%, 10%, 5% or 1% of the ESM components will show evidence of degradation as compared to naturally occurring ESM from a corresponding avian source. Non-digested, non-reduced and non-denatured should be interpreted accordingly. The degree of degradation/digestion/reduction/denaturation of ESM can be assessed by measuring the relative size or structure of the collagen fibres in the ESM and/or the extent of collagen cross-linking in the ESM and/or the extent of disulphide bonding. This may be achieved through routine techniques including immunohistochemistry/immunocytochemistry techniques and/or biomolecule (e.g. protein) stains and dyes and or detecting sulphide aromas.

Nevertheless, in some embodiments, the ESM to be incubated with the strong base may washed during its isolation with an acid solution of pH 0.8 to 1.5, e.g. 1 to 1.2. Acids including hydrochloric acid, citric, nitric, phosphoric, carbonic or acetic acid may be used. This treatment is performed under conditions suitable for the demineralisation of the ESM, thus minimising the amount of inorganic salts (e.g. calcium carbonate) in the ESM, and/or for the removal and/or inactivation of infective agents, e.g. microorganisms (e.g. as described herein), prions and viruses. Conditions should be selected to avoid or minimise hydrolysis and/or reduction of the ESM components.

Eggshell membrane may be separated from other egg components by any convenient means. The eggs from which the eggshell membrane may be separated may be fertilised or unfertilised. The eggs may be intact, i.e. prior to hatching, or may be empty, i.e. the remnants of the egg following hatching or following extraction of the egg contents (albumen and yolk). Suitable means are for example described in WO 2004/080428 and US 8580315, the contents of which are incorporated herein by reference. Preferably the ESM is prepared by the method for harvesting eggshell membrane in-line in commercial egg processing plants -7 -disclosed in WO 2015/058790 (PCT/EP2013/072049) the contents of which are incorporated herein by reference.

WO 2015/058790 provides a method of processing eggshell residues, which emanate from an egg breaking unit and comprise eggshell portions as well as membrane portions, comprising feeding eggshell residues (e.g. having a particle size of about 0.5mm to about 40mm and a wet basis moisture content of about 3% to about 40%) from the egg breaking unit into a cyclone driven by a process gas having a temperature of less than about 85°C (preferably of less than about 60°C) and having a speed exceeding about 60m/s (preferably between about 70 m/s and about 340 m/s). Within said cyclone vortex processing of the eggshell residues reduces particle size and peels said membrane portions off of said eggshell portions, such that said eggshell portions become separated from said membrane portions. Through a top outlet of said cyclone there is released mainly a mix of process gas, vapour and droplets, and through a bottom outlet of said cyclone there is released mainly a mixture of separated eggshell portions and membrane portions. Said released mixture is then separated into an eggshell portion part and a membrane portion part in a sorting device. The resultant ESM portion (typically flaked ESM) may be used in the invention as it is in wet or dry form, or may then be processed further into particulate ESM and ESM particles in wet or dry form. It may be especially advantageous to use the wet pulp of flaked ESM obtained directly from this process as the insoluble ESM starting material. The use of this comparatively unprocessed product may result in time, cost and energy savings.

Flaked ESM is considered to have a size range of around 1mm2 to about 10mm2.

In accordance with the invention the term "particulate ESM" may be a, or may be formed from at least one, particle of ESM having a mean particle diameter of up to 1mm, e.g. up to 500,450, 400, 350, 300, 250, 200, 150, 125 or 100 pm. In certain embodiments particulate ESM may be a, or may be formed from at least one, particle of ESM having a mean particle diameter of less than 100 pm, e.g. less than 200, 150, 100, 50, 10, 5 or 1 nm.

95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or 1 pm, e.g. less than 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, In certain other embodiments particulate ESM may be a, or may be formed from at least one, particle of ESM having a mean particle diameter of equal to or greater -8 -than 1 nm, e.g. equal to or greater than 5, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 950 nm, or equal to or greater than 1 pm, e.g. equal to or greater than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 250, 300, 350, 400, 450 or 500 pm.

Any and all range endpoints derivable from the combination of any of these values are specifically contemplated.

ESM particles may be any shape. An ESM particle may be essentially symmetric or asymmetric. An ESM particle may be essentially spherical, prismatoidal or cylindrical. An ESM particle may be essentially irregular or regular or have regions of both. An ESM particle may be angular, rounded or tapered or have regions thereof In certain embodiments an ESM particle may have one length dimension that is significantly greater than the others and so may be referred to as, for example, rod-shaped, needle-shaped or fibrous (rods, needles or fibres) and may be qualified as cylindrical or prismatoidal (e.g. cuboidal) depending on the cross-sectional shape substantial perpendicular to the dimension of significantly greater length.

In view of the generality of the invention with regard to ESM particle shape, in the context of ESM particles which are not substantially, e.g. essentially, spherical, references to ESM particle diameters are therefore references to equivalent spherical diameter. In these embodiments the ESM particle has a shape defined by size dimensions that would result in the same size readings as a sphere of the same substance composition of said diameter in the particle size measuring technique used. In certain embodiments the size dimensions used are volume or surface area, preferably volume. Thus, in accordance with the invention ESM particles with irregular shapes may have an equivalent spherical diameter of up to 1 pm, e.g. up to 500, 450, 400, 350, 300, 250, 200, 150, 125 or 100 pm. In certain embodiments ESM particles with irregular shapes may have an equivalent spherical diameter of less than 100 pm, e.g. less than 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or 1 pm, e.g. less than 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 10, 5 or 1 nm. In particular embodiments ESM particles with irregular shapes may have an -9 -equivalent spherical diameter of less than 80 pm, 70 pm, 65 pm, 60 pm, 55 pm, 50 pm, 45 pm, 40 pm, 35 pm, or 30 pm.

In certain other embodiments ESM particles with irregular shapes may have an equivalent spherical diameter of equal to or greater than 1 nm, e.g. equal to or greater than 5, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 950 nm, or equal to or greater than 1 pm, e.g. equal to or greater than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 250, 300, 350, 400, 450 or 500 pm.

The mean (average) diameter, or equivalent spherical diameter, may be assessed by any convenient means, e.g. resistive pulse/Coulter method, sedimentation (gravity or centrifugation), optical imaging (e.g. SEM, static image analysis, dynamic image analysis), laser diffraction or light scattering, but for the purposes of the invention the Coulter method, in the form of Tunable Resistive Pulse Sensing, or optical means should be used to determine particle size.

The particulate ESM of use in the invention, and thus the ESM particles which make up particulate ESM, may be prepared from ESM, e.g. flakes, shreds or sheets, by any convenient particle size reduction, micronizing, grinding, pulverizing or milling technology means, e.g. ball milling, bead milling, jet milling, vortex milling, blade milling, rotor-stator dispersement, optionally followed by size selection, e.g. sieving and screening. The chosen particle size reduction method may be either performed dry or with a liquid medium. Cryo-pulverization may also be employed.

The method of the invention may further comprise preceding steps of preparing the insoluble ESM of use in the invention, e.g. as described specifically above.

In accordance with the invention it has been found that an aqueous solution of a strong base may be used at high temperature and elevated pressure to rapidly hydrolyse high concentrations of water insoluble ESM into soluble peptides without disrupting disulphide bridges and thereby producing unpleasant sulphide odours.

The peptides are of varying sizes and are expected to have bioactive properties rendering them useful in various industrial applications.

-10 -The aqueous mixture of the ESM and the strong base may be formed in any convenient way. The ESM (e.g. in aqueous suspension or in solid form) may be added to an aqueous solution of the base, an aqueous solution of the strong base may be added to the ESM (e.g. in aqueous suspension or in solid form), solid base may be added to an aqueous suspension of the ESM, an aqueous liquid may be added to a mixture of ESM and solid base, or any combination of these approaches may be used. In all instances the resultant aqueous mixture should have an initial pH of at least 13.0, e.g. at least 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9 or 14.0. An initial pH of 13.0 to 14.0, e.g. 13.5 to 14.0, may be selected. Without wishing to be bound by theory, in the method of the present invention hydrolysis of the insoluble ESM material does not occur at appreciable levels at a pH below 13.0. As such, in certain embodiments, additional base, e.g. in liquid or solid form, is added to the aqueous mixture during the incubation to maintain the pH at at least 13.0, e.g. at least 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9 or 14.0, e.g. 13.0 to 14.0 or 13.5 to 14.0 for at least part of the incubation. In other embodiments it may be advantageous to allow the pH to decrease over all, or the latter part, of the incubation to control the extent of hydrolysis. In some embodiments the pH of the reaction will become less than 13.0, e.g. equal to or less than 12.5, 12.0, 11.5, 11.0, 10.5, or 10.0, during the hydrolysis reaction, thereby ceasing hydrolysis of the ESM before it is complete.

In certain embodiments, the skilled person would be able to select an amount of ESM starting material and an amount of base such that the starting pH will be at least 13.0, e.g. at least 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9 or 14.0, e.g. 13.0 to 14.0 or 13.5 to 14.0 and that the pH will become less than 13.0, e.g. equal to or less than 12.5, 12.0, 11.5, 11.0, 10.5, or 10.0, before all of the ESM starting material will be hydrolysed to amino acids and/or disulphide bonds are reduced, thereby resulting in a aqueous peptide-containing hydrolysis product (aqueous solution of water soluble ESM peptides) which are predominantly oligopeptides with intact disulphide bonds.

In other embodiments hydrolysis may be stopped at any desired time through the addition of an amount of acid sufficient to lower the pH to less than 13.0, e.g. equal to or less than 12.5, 12.0, 11.5, 11.0, 10.5, or 10.0.

The term "aqueous" is intended to mean that a liquid composition to which it relates has a liquid portion which is comprised substantially, e.g. predominantly or essentially, of water, e.g. at least 80%, 90%, 95%, 99% or 100% of the liquid portion of the entity is water. In other embodiments less than 20%, 10%, 5%, or 1% of the liquid portion is a non-polar solvent. Solvents with a dielectric constant of less than 15 are generally considered to be non-polar. Expressed differently an aqueous liquid composition can be considered to comprise less than 20%, 10%, 5%, or 1% of a non-polar organic liquid phase. Preferably an aqueous liquid composition in accordance with the invention is devoid of a non-polar liquid phase.

It is an advantage of the invention that the method is capable of hydrolysing large amounts of insoluble ESM starting material into constituent water soluble peptides and thus forming an aqueous solution of water soluble eggshell membrane peptides of correspondingly high concentration (i.e. an aqueous solution which is highly enriched for the water soluble eggshell membrane peptides). Thus, if the ESM content of the aqueous mixture is suitably high, a separate step of concentrating the aqueous solution of water soluble eggshell membrane peptides, e.g. by dialysis and the like, is not necessary. Such steps add complexity and costs and reduce yields. In certain embodiments the aqueous mixture of a strong base and water insoluble eggshell membrane contains ESM at equal to or greater than 25% w/v, e.g. equal to or greater than 30%, 35%, 40%, 45%, 50%, 55%, or 60% w/v. The proportion of ESM starting material may in certain embodiments be limited by the form in which it is provided for hydrolysis. Typically, the smaller the fragment size of the ESM, the greater the amount of ESM which may be incorporated in the hydrolysis reaction. Without wishing to be bound by theory, it is believed that the smaller the fragment size the greater is its capacity to be wetted (proportional to surface area) and so available for hydrolysis. If a high concentration of ESM peptides in the final product is desired it may be necessary to select a smaller insoluble ESM fragment size.

In other embodiments the above amounts are starting amounts and the hydrolysis reaction may be supplemented with additional ESM (and additional base if necessary) as the reaction progresses in order to increase the amount or concentration of water soluble eggshell membrane peptides in the reaction product.

The strong base may be any base which can be formulated as an aqueous solution with the necessary pH. Examples include, but are not limited to sodium hydroxide, -12 -potassium hydroxide, calcium hydroxide, barium hydroxide, strontium hydroxide, or combinations thereof. The strong base may be selected to result in a specific ion profile in the aqueous solution of water soluble eggshell membrane peptides and thus final peptide product. In certain embodiments the base is sodium hydroxide and/or potassium hydroxide.

It will be readily appreciated by those skilled in the art that the time and temperature required for the hydrolysis step to take place are interconnected variables and that, in turn, the chosen pH of the mixture and the pressure at which the incubation is performed would also impact on these variables. The amount of ESM in the starting mixture is a still further variable which may affect the time and temperature used. Moreover, the extent to which the protein components in the ESM are to be hydrolysed (i.e. the size of the water soluble peptides the skilled person seeks to obtain) is a further factor which can affect the choice of time and temperature.

Thus, within the limits specified above, incubation of the aqueous mixture of the strong base and the water insoluble ESM is for a time and at a temperature and pressure sufficient for hydrolysis of the insoluble ESM to occur and thereby form an aqueous solution of (highly enriched) water soluble eggshell membrane peptides. Complete hydrolysis of the insoluble ESM starting material is not essential as non-hydrolysed insoluble ESM will be removed in the later steps.

In certain embodiments temperatures of about 111 °C to about 130 °C, preferably about 11213, 11313, 11413, 11513, 11613, 11713, 11813, 11913, 12013, 121 °C, 122 °C, 12313, 12413, 12513, 12613, 12713, 12813, or 12913, to about 130 °C, or about 112 °C to about 113 °C, 114 °C, 115 °C, 116 °C, 117 °C, 118 °C, 119 °C, 120 °C, 121 °C, 122 °C, 123 °C, 124 °C, 12513, 12613, 12713, 128 °C, or 129 °C may be appropriate. Any and all range endpoints derivable from the combination of any of these values are specifically contemplated. In certain embodiments a temperature range of 120 °C to 125 °C, e.g. about 121 °C may be appropriate.

The incubation step may be run at different temperatures from time to time, e.g. two or more of the above mentioned temperatures, for example to control the rate of hydrolysis, the structure of the peptides so obtained or to achieve energy or cost efficiencies.

-13 -In certain embodiments, pressures of at least 155 kPa, e.g. at least, 160 kPa, 165 kPa, 170 kPa, 175 kPa, 180 kPa, 185 kPa, 190 kPa, 195 kPa, 200 kPa, 205 kPa, 210 kPa, 215 kPa, 220 kPa, 225 kPa, 230 kPa, 235 kPa, 240 kPa, 245 kPa, 250 kPa, 255 kPa, 260 kPa, 265 kPa, or at least 270 kPa may be appropriate. The pressure selected may influence the time and pH needed to achieve appropriate hydrolysis. In certain embodiments the hydrolysis reaction is performed at a pressure of approximately 215kPa, e.g. 150 to 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, or 270 kPa, or 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, or 260 to 270 kPa. Any and all range endpoints derivable from the combination of any of these values are specifically contemplated.

Any suitable means or technique known in the art can be used for maintaining the pressure during the incubation step. For example, the required pressure can be achieved and maintained using an autoclave (e.g. a stovetop autoclave or pressure cooker, a horizontal high-capacity autoclave or pass-through autoclave, etc.). The technique of pascalization may also be used to achieve the required pressures for the incubation step of the present method. Further approaches include steam pasteurisation, e.g. direct steam injection pasteurisation, and ultra-high temperature processing.

In certain embodiments the aqueous mixture may be maintained at the incubation temperature and pressure for about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 minutes to about 30 minutes, or for about 12 to about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 minutes.

Incubation for these times represents a rapid hydrolysis step.

The hydrolysis reaction of the present reaction may involve a plurality of incubation steps if that would be convenient or advantageous to the process and/or the final product. This may provide control over the rate of hydrolysis, the structure of the peptides so obtained or offer energy or cost efficiencies. Each of the plurality of steps may be of a time recited above, or may total a time recited above.

The extent to which the hydrolysis reaction is allowed to proceed may vary, e.g. depending on the intended application for the final peptide product. Indeed, it will typically be the case that hydrolysis is not allowed to proceed to completion, i.e. -14 -complete breakdown of the ESM proteins into their constituent amino acids because peptides are likely to have a wider range of potential applications. However, in certain embodiments hydrolysis may be allowed to proceed to such an extent that high levels of amino acids are present in the final peptide product.

Progress of the hydrolysis reaction may be monitored by any convenient means.

Many routine options are available to the skilled person. For example, the progress of the hydrolysis step may be monitored by taking samples, e.g. over regular time intervals, and analysing said samples by spectrophotometry, e.g. UV spectrophotometry. Soluble proteins are detected directly and can be quantified at wavelengths of, for example, 214 and 280 nm. In other approaches, colorimetric methods (e.g. Bradford, Lowry, Biuret, BOA, etc.) in which the absorption of light by specific protein binding dyes are used to indirectly detect proteins in solution, may be used. In other embodiments mass spectroscopy (e.g. using matrix-assisted laser desorption/ionization (MALDI) time of flight apparatus) may be used to characterise the peptide products, including their amino acid composition and sequence. Still, further investigation into the hydrolysis step can utilise, for example, SDS-PAGE or HPLC techniques. SDS-PAGE can be performed under reducing and non-reducing conditions to assess the status of disulphide bonds.

Thus, techniques well-known in the art would readily allow the skilled person to determine whether more time is required to perform the hydrolysis step, or alternatively, whether the reaction has proceeded too long as indicated by hydrolysis of essentially all of the eggshell membrane proteins into their constituent amino acids. In certain embodiments is may be advantageous for the hydrolysis reaction to not proceed as far as breaking the ESM protein components down into amino acids.

The method of the present invention includes a step of separating insoluble residues from the alkaline hydrolysate, i.e. the aqueous solution of water soluble eggshell membrane peptides formed by the alkaline hydrolysis reaction.

Separation of insoluble residues from the peptide solution should not be taken as implying that the insoluble residue must be removed from the peptide solution and thus encompasses techniques in which the peptide solution is removed from the insoluble residue. Such residues may be, for instance, fragments of eggshell, fine calcium or other mineral deposits from eggshell, aggregates of organic molecules and non-hydrolysed fragments of ESM.

-15 -Many suitable separation techniques are available and would be routine for the skilled person to employ. For instance, the skilled person would have no trouble applying size filtration-based techniques by selecting filters or gels with appropriately sized pores, or chromatography columns with appropriately sized particulate solid supports, centrifugation-based techniques, and gravity-based techniques (allowing the liquid preparation to stand for sufficient length of time for the insoluble residues to settle). A filtration step may be most convenient. There are a number of filtration techniques known in the art including, but not limited to, simple (gravity) filtration, hot or cold filtration, vacuum filtration, membrane filtration, batch filtration, pressure filtration, continuous filtration. A suitable combination thereof may be used.

Filters with a cut-off size of 1 pm, e.g. 0.95, 0.90, 0.85, 0.80, 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10, or 0.05 pm, may be suitable. Conveniently filters with a cut-off size of 0.45 pm or less, e.g. 0.22 or 0.20 pm may be used because these filters will also remove cellular microorganisms. In certain embodiments a series of filters of the same size or descending size may be used, e.g. to remove progressively smaller particles so as to avoid clogging. Suitable filters for this step, e.g. in the form of cartridge systems, are commercially available.

The separation step may conveniently be performed at 25 °C, but other temperatures may be used so long as compatible with the separation technique being used. A lower temperature may be used to ensure further hydrolysis does not occur. The pH of the peptide solution on which the separation step is performed should be maintained at equal to or less than 12.1, e.g. equal to or less than 12.0, 11.5, 11.0, 10.5, or 10.0, to prevent further hydrolysis.

The residue-depleted solution recovered by this separation step is then neutralised, i.e. acidified to reduce the pH of the solution to a neutral pH. In certain embodiments, the acidification step reduces pH of the solution to a pH of 6 to 8, e.g. 6.4 to 7.6, 6.5 to 7.5, 6.6 to 7.4, 6.7 to 7.3, 6.8 to 7.2, preferably to a pH of about 7.0. In other embodiments the residue-depleted solution may be neutralised to a physiological pH, e.g. pH 7.3 to 7.9, 7.4 to 7.8, 7.5 to 7.7, or about 7.6. The neutralisation step may conveniently be performed at 25 °C, but other temperatures -16 -may be used. For example, a lower temperature may be used to help maintain peptide stability.

This neutralisation step gives the peptide solution wider utility because it may be added to other products or formulated into products without affecting the pH of those products to any significant extent. This is of particular importance for uses in therapeutic, nutritional and cosmetic applications and in in vitro biological contexts, e.g. in cell, tissue and organ culture systems.

Suitable acids for the neutralisation of the residue-depleted peptide solution include, but are not limited to, hydrochloric, sulphuric, citric, acetic, ascorbic, benzoic, lactic, oxalic, phosphoric, chloroacetic, formic, succinic, propionic, nitric, perchloric, or chloric acid. Neutralisation of the basic residue-depleted peptide solution with an acid will create salts of the base and the acid counter ions. The acid, or combination thereof, used may therefore be selected to result in the formation of salts which would be beneficial to the final peptide preparation, e.g. to improve peptide stability and shelf-life or ease of formulation, or which would make the final peptide preparation physiologically acceptable or beneficial to the intended recipient. The choice of acid used at this point may be influenced by, or influence, the choice of base used in the hydrolysis step. Preferably, hydrochloric and/or citric acid is used so as to form salts routinely used in pharmaceutical, nutraceufical and cosmetic compositions.

The method of the present invention may further comprise a dilution step after the neutralisation step (step (d)) which brings the conductivity of the neutralised peptide solution to a physiological range of conductivity. This step may be present where the final peptide preparation is intended for administration to human or animal subjects or for use in cell and tissue culture contexts. Where the final peptide preparation is destined for use in other contexts this step might not be present. In certain embodiments, the starting insoluble ESM material may be provided as a wet pulp or other wetted form and, in such embodiments, the volume of water that is held up in the pulp or provided with the ESM may, or could be designed to, result in an aqueous solution of water soluble ESM peptides which would be at a physiological conductivity upon neutralisation. In such embodiments, a dilution step would not be required.

-17 -In accordance with the present invention, a physiological range of conductivity for an aqueous solution is a conductivity of 8 to 14 milliSiemens/cm (mS/cm), e.g. 8, 9, 10, 11, 12 or 13 to 14mS/cm or 8 to 9, 10, 11, 12, 13 or 14mS/cm. Any and all range endpoints derivable from the combination of any of these values are specifically contemplated. In a preferred embodiment, the dilution step alters the conductivity of the neutralised peptide solution to 10 to 12 ms/cm. Conductivity of aqueous liquids may be measured by routine means such as commercially available electrical conductivity meters. The above figures are given for a solution at 25 °C.

In further embodiments the method comprises a desalting step, e.g. by gel filtration chromatography or dialysis. In certain embodiments this step takes place after the neutralisation step (step (d)).

Dilution may be achieved by combining the neutralised peptide solution with an aqueous liquid of lower conductivity. To avoid over dilution of the soluble peptides, the diluent should have significantly lower conductivity, and so is preferably water.

In certain embodiments the water is purified water. As used herein, the term "purified water" refers to water that has been mechanically filtered or processed to remove impurities. Standard methods for purifying water are well-known in the art, including, but not limited to, distillation, capacitive deionizafion, reverse osmosis, carbon filtering, microfiltration, ultrafiltration, ultraviolet oxidation, or electrodeionization, or suitable combinations thereof. The diluent may be sterile filtered prior to combining with the neutralised peptide solution.

Optionally the method of the invention may comprise a step in which the neutralised residue-depleted peptide solution or the diluted neutralised residue-depleted peptide solution is filtered to remove cellular microorganisms (e.g. bacteria, fungi, algae, protozoa and the like). This may be referred to as "sterile filtration" and is a process common in the field of preparing sterile compositions for administration to human or animal subjects or for use in cell and tissue culture contexts. It is generally considered that filtration of a liquid using a filter with a pore size of 0.45 pm or less, preferably 0.22 pm or less, or 0.2 pm or less, will remove essentially all cellular microorganisms and render the filtered liquid sterile. Thus, in certain embodiments the neutralised residue-depleted peptide solution or the diluted neutralised residue-depleted peptide solution is filtered with a filter with a pore size -18 -of 0.45 pm or less, preferably 0.22 pm or less, or 0.2 pm or less. In certain embodiments filtration of the neutralised residue-depleted peptide solution or the diluted neutralised residue-depleted peptide solution is performed immediately prior to sterile packaging of the final peptide preparation, e.g. in sterile glass containers.

In certain embodiments one or more additional physical sterilisation steps are included in the method. Such sterilisation techniques may include irradiation (e.g. UV), high pressure, and nanofiltration (virus filtration) and/or additional sterile filtration steps as described above. In certain embodiments such steps are performed immediately prior to sterile packaging of the final peptide preparation, but they may be performed at other junctures of the method if desired.

As discussed previously, the method of the invention may be performed such that the presence of sulfhydryl (thiol) groups in the final peptide product is minimised so as to minimise malodours (rotten egg aromas) associated with the reduction of the disulphide bridges present in the ESM protein structures. In preferred embodiments such groups will be substantially absent, or at least present in amounts no greater than in the eggshell membrane starting material. In most preferred embodiments such groups will be essentially absent, i.e. undetectable by routine means. Viewed alternatively, the number of disulphide bonds in the peptide solution will be essentially the same, or no less than that in the eggshell membrane starting material. Quantification of thiols and disulphides may be performed by any convenient means, e.g. as described in Wnther and Thorpe, 2014, Biochimica et Biophysica Acta (BBA) -General Subjects, Vol 1840(2), 838-846, or by the detection of rotten egg aromas by nose or artificial means. In these embodiments the treatment of the final peptide product, or any intermediate solution described herein with odour-absorbing or odour-reducing compounds is not required. Thus, in certain embodiments the present invention does not include a step in which the final peptide product, or any intermediate solution described herein is treated with odour-absorbing or odour-reducing compounds, e.g. charcoal and hydrogen peroxide Nevertheless, in other embodiments the treatment of the final peptide product, or any intermediate solution described herein with odour-absorbing or odour-reducing compounds is not excluded.

In other embodiments, the method of the present invention may further comprise a step in which the final peptide solution 0.e. the neutralised residue-depleted peptide -19 -solution, the diluted neutralised residue-depleted peptide solution or the sterile filtered forms thereof) is dried, thereby forming a dry soluble eggshell membrane peptide composition.

The dry soluble eggshell membrane peptide composition is substantially, e.g. essentially, water-free (moisture-free). This may be expressed as a water content of less than 10% w/w, e.g. less than 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5% or 1% w/w as measured by weight loss on drying or chemically by the Karl Fischer method (United States Pharmacopeia; European Pharmacopoeia).

The drying of the final peptide solution can be performed using any suitable drying technique. These techniques include, but are not limited to, lyophilisation or freeze drying, natural air drying, vacuum drying, paddle drying, drum drying, supercritical drying, or a suitable combination thereof. In a preferred embodiment, the drying step is performed by lyophilisation. The drying step may be performed at any suitable temperature and it will be appreciated that the temperature will vary based on the drying technique chosen. With lyophilisation, for example, a minimum temperature in the range of about -30 °C to -80 °C may be used in the primary drying phase. Lyophilisation may further comprise a secondary drying phase where the temperature is raised higher than in the primary phase to, for example, temperatures greater than 0 °C. In certain embodiments the final peptide solution is frozen to about -40 °C with primary drying taking place over a shift from -40 °C to 0 °C and secondary drying taking place at 20 °C.

In certain embodiments the dry composition will preferably be in the form of a powder. A common problem when forming powders is caking. As used herein, the term "caking" refers to the tendency of a powder to form lumps or masses (i.e aggregates). The formation of such masses or lumps interferes with packaging, transport, flowability, and consumption of the powder. Advantageously, it has been found that the soluble peptide solution of the present invention can be readily dried to a powder with no such problems, e.g. using the above described methods. Nevertheless, should aggregates of an undesirable size form during the process, these may be broken down with any convenient size reduction technique, e.g. those described above for preparing ESM particles.

The dry soluble eggshell membrane peptide composition may undergo a physical sterilisation prior to or following packaging, e.g. into sterile glass containers.

-20 -In a further aspect, the present invention provides a method for preparing water soluble eggshell membrane peptides, wherein said method comprises: (a) incubating an aqueous mixture of a strong base and water insoluble eggshell membrane at a pH of at least 12.2 for a time and at a temperature sufficient for hydrolysis of the eggshell membrane to occur and thereby form an aqueous solution of water soluble eggshell membrane peptides; (b) separating insoluble residues from the peptide solution formed in step (a); and (c) neutralising the residue-depleted peptide solution; and optionally (d) diluting the neutralised residue-depleted peptide solution with an aqueous liquid to a physiological conductivity, optionally wherein the neutralised residue-depleted peptide solution or the diluted neutralised residue-depleted peptide solution is filtered to remove cellular microorganisms.

In this aspect, the aqueous mixture should have an initial pH of at least 12.2, e.g. at least 12.3, 12.4, 12.5, 12.6, 12.7, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9 or 14.0. An initial pH of 13.0 to 14.0, e.g. 13.5 to 14.0, may be selected. In certain embodiments additional base, e.g. in liquid or solid form, is added to the aqueous mixture during the incubation to maintain the pH at at least 12.2, e.g. at least 12.3, 12.4, 12.5, 12.6, 12.7, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9 or 14.0, e.g. 13.0 to 14.0 or 13.5 to 14.0 for at least part of the incubation. In other embodiments it may be advantageous to allow the pH to decrease over all, or the latter part, of the incubation to control the extent of hydrolysis. In some embodiments the pH of the reaction will become equal to or less than 12.1, e.g. equal to or less than 12.0, 11.5, 11.0, 10.5, or 10.0, during the hydrolysis reaction, thereby ceasing hydrolysis before it is complete.

In certain embodiments of this aspect, the skilled person would be able to select an amount of ESM starting material and an amount of base such that the starting pH will be at least 12.2, e.g. at least 12.3, 12.4, 12.5, 12.6, 12.7, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9 or 14.0, e.g. 13.0 to 14.0 or 13.5 to 14.0 and that the pH will become equal to or less than 12.1, e.g. equal to or less than 12.0, 11.5, 11.0, 10.5, or 10.0, before all of the ESM starting material will be -21 -hydrolysed to amino acids and/or disulphide bonds are reduced, thereby resulting in a aqueous peptide-containing hydrolysis product (aqueous solution of water soluble ESM peptides) which are oligopeptides with intact disulphide bonds.

In other embodiments of this aspect, hydrolysis may be stopped at any desired time thorough the addition of an amount of acid sufficient to lower the pH to equal to or less than 12.1, e.g. equal to or less than 12.0, 11.5, 11.0, 10.5, or 10.0.

In certain embodiments of this aspect, the aqueous mixture of a strong base and water insoluble eggshell membrane contains ESM at equal to or greater than 10% w/v, e.g. equal to or greater than 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% w/v.

In certain embodiments of this aspect, temperatures of at least 100 C, e.g. at least 105 °C, 110 °C, 115 °C, 120 °C, 125 °C, 130 °C, 135 °C, 140 °C, 145 °C or at least 150 °C may be appropriate. In certain embodiments temperatures of about 100 °C to about 150 °C, preferably about 105 °C, 110 °C, 115 °C, 120 °C, 125 °C, 130 °C, 135 °C, 140 °C, or 145 °C to about 150 °C, or about 100 °C to about 105 °C, 110 °C, °C, 120 °C, 125 °C, 130 °C, 135 °C, 140 °C, or about 145 °C may be appropriate. Any and all range endpoints derivable from the combination of any of these values are specifically contemplated. In certain embodiments a temperature range of 120 °C to 125 °C, e.g. about 121 °C may be appropriate.

In certain embodiments of this aspect the aqueous mixture may be maintained at the incubation temperature for a period ranging from minutes to hours, for example, for at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or at least 120 minutes. In certain embodiments, the aqueous mixture may be maintained at the incubation temperature for a period ranging from about 10 to about 120 minutes, e.g. from about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or about 110 minutes to about 120 minutes, or about 10 minutes to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or about 110 minutes. Any and all range endpoints derivable from the combination of any of these values are specifically contemplated. In preferred embodiments the aqueous mixture may be maintained at the incubation temperature or pressure for about 10 to about 30 minutes, preferably about 30 minutes. Incubation for these times represents a rapid hydrolysis step.

In certain embodiments of this aspect, the hydrolysis step may be run at any suitable pressure. The pressure selected may influence the time and pH needed to -22 -achieve appropriate hydrolysis. In certain embodiments the hydrolysis reaction is performed at a pressure of approximately 215kPa, e.g. 150 to 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, or 270 kPa, or 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, or 260 to 270 kPa. Any and all range endpoints derivable from the combination of any of these values are specifically contemplated.

In a further aspect the invention provides water soluble eggshell membrane peptides obtained or obtainable from the methods described above. All specific embodiments described above in any and all combinations apply mutatis mutandis to this aspect.

Thus, in this aspect the peptides may, for example, be in the form of a solution or a dry solid, e.g. as described above. As such, expressed alternatively, the invention provides a solution of water soluble eggshell membrane peptides obtained or obtainable from the methods described above and a dry solid mixture of water soluble eggshell membrane peptides obtained or obtainable from the methods described above.

In a further aspect the invention provides a composition comprising water soluble eggshell membrane peptides obtained or obtainable from the methods described above. The composition may, for example, be in liquid or solid form as appropriate for its intended use.

The composition may be a therapeutic (e.g. pharmaceutical or nutraceufical), cosmetic, or nutritional composition. The composition may alternatively be designed to meet the requirements of use in in vitro biological contexts, e.g. in cell, tissue and organ culture systems. These compositions will further comprise carriers, diluents or excipients appropriate for said use.

In the context of therapeutic treatments, the ESM peptides of the invention may be administered to a subject in any convenient form or by any convenient means, e.g. by topical, oral, enteral or parenteral routes, or by inhalation. Parenteral routes include intrathecal, intramuscular and intravenous routes. In preferred embodiments, the pharmaceutical composition is administered orally or topically.

The skilled person will be able to formulate the ESM peptides of the invention into pharmaceutical compositions that are adapted for the above described routes of administration according to any of the conventional methods known in the art and widely described in the literature.

-23 -Thus, in one embodiment there is provided a pharmaceutical composition comprising water soluble eggshell membrane peptides obtained or obtainable from the methods described above together with at least one pharmaceutically acceptable carrier, diluent or excipient. This composition may also comprise other therapeutic agents In some embodiments the pharmaceutical composition may be referred to as a nutraceutical on account of the source of the peptides. The following may be interpreted accordingly.

The pharmaceutical compositions of the invention can be applied to any affected part of a subject's body. It will be appreciated that the pharmaceutical composition of the present invention may be applied alone or in combination with another form of treatment or treatments. The pharmaceutical composition described herein may be administered one time or multiple times, depending on the disease, the disease state and severity of the disease. For example, the composition may be administered 1, 2 or more times per day and for 1, 2 or more consecutive or alternating days. Such compositions may be applied for a prescribed period of time, or indefinitely depending on the disease in question. Dosage amount and interval may be adjusted accordingly to provide the correct amount of the pharmaceutical composition which is sufficient to achieve a desired effect. One skilled in the art will readily be able to formulate and optimise the appropriate dosage without undue burden.

More specifically, the ESM peptides of the invention may be incorporated, optionally together with other active agents, with one or more conventional carriers, diluents and/or excipients, to produce conventional galenic preparations such as tablets, pills, powders (e.g. inhalable powders), lozenges, sachets, cachets, elixirs, suspensions, emulsions, creams, gels, foams, solutions, syrups, aerosols (as a solid or in a liquid medium), sprays (e.g. nasal sprays), compositions for use in nebulisers, ointments, capsules (e.g. soft and hard gelatine), suppositories, pessaries, sterile injectable solutions, sterile packaged powders, and the like.

Sterile injectable compositions are of particular note.

In one embodiment, the pharmaceutical composition is administered topically in the form of a cream, gel or foam. In other embodiments, the pharmaceutical composition is administered orally in the form of a capsule or tablet. In further -24 -embodiments, the pharmaceutical composition is administered as a sterile solution via an injection.

Examples of suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, inert alginate polymers, tragacanth, gelatine, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, water, water/ethanol, water/ glycol, water/polyethylene, hypertonic salt water, glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof.

Excipients and diluents of note are mannitol and hypertonic salt water (saline).

The compositions may additionally include lubricating agents, binding agents, glidants (flow aids), wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, colouring agents and the like. Additional therapeutically active agents may be included in the pharmaceutical compositions.

Parenterally administrable forms, e.g., intravenous solutions, should be sterile and free from physiologically unacceptable agents, and should have low osmolarity to minimize irritation or other adverse effects upon administration and thus solutions should preferably be isotonic or slightly hypertonic, e.g. hypertonic salt water (saline). Suitable vehicles include aqueous vehicles customarily used for administering parenteral solutions such as sterile water for injection, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection and other solutions such as are described in Remington's Pharmaceutical Sciences, 15th ed., Easton: Mack Publishing Co., pp. 1405-1412 and 1461-1487 (1975) and The National Formulary XIV, 14th ed. Washington: American Pharmaceutical Association (1975). The solutions can contain preservatives, antimicrobial agents, buffers and antioxidants conventionally used for parenteral solutions, excipients and other additives which are compatible with the biopolymers and which will not interfere with the manufacture, storage or use of products.

For topical administration the ESM peptides of the invention can be incorporated into creams, foams, ointments, gels, transdermal patches and the like. The ESM peptides can also be incorporated into medical dressings, for example wound -25 -dressings e.g. woven (e.g. fabric) dressings or non-woven dressings (e.g. gels or dressings with a gel component).

Further topical systems that are envisaged to be suitable are in situ drug delivery systems, for example gels where solid, semi-solid, amorphous or liquid crystalline gel matrices are formed in situ and which may comprise the ESM peptides of the invention. Such matrices can conveniently be designed to control the release of the ESM peptides from the matrix, e.g. release can be delayed and/or sustained over a chosen period of time. Such systems may form gels only upon contact with biological tissues or fluids. Typically the gels are bioadhesive. Delivery to any body site that can retain or be adapted to retain the pre-gel composition can be targeted by such a delivery technique.

The relative content of the ESM peptides in the compositions of the invention can vary depending on the dosage required and the dosage regime being followed and this will depend on the subject to be treated and the target disease or condition.

Preferably, the composition will comprise an amount of ESM peptides that will provide measurable, preferably clinically relevant, prevention or treatment of the target disease.

In other embodiments the ESM peptides of the invention may be present as excipients for pharmaceutically active agents. In particular, the ESM peptides of the invention may be used to prepare gel, e.g. hydrogel, based formulations, e.g. those described above, for pharmaceutically active agents.

The pharmaceutical compositions of the invention will typically comprise 1% to 99%, 5% to 95%, 10% to 90% or 25% to 75% of the ESM peptides of the invention, allowance being made for other ingredients.

In another aspect, the invention provides the pharmaceutical composition of the present invention for use in therapy or for use as a medicament in the treatment or prevention of one of more diseases or conditions treatable or preventable with the ESM peptides of the invention.

Expressed differently, the invention provides the ESM peptides of the invention for use in therapy or for use as a medicament in the treatment or prevention of a disease or condition treatable or preventable with the ESM peptides of the invention.

-26 -Thus, the invention further provides a method for the treatment or prevention of a disease or condition treatable or preventable with the ESM peptides of the invention, said method comprising administering the ESM peptides of the invention or a composition comprising the ESM peptides of the invention to a subject in need thereof.

More specifically, the ESM peptides of the invention and pharmaceutical compositions comprising the same may be provided for use in the treatment of diseases or conditions including, but not limited to, autoimmune diseases (e.g. rheumatoid arthritis, diabetes, etc.), osteoarthritis, inflammatory diseases, gastrointestinal disorders (e.g. colitis, irritable bowel syndrome, etc.), hypertension, myocardial infarction, stroke, heart failure, joint inflammation and pain. The salts present in the ESM peptide preparations of the invention should be selected depending on the disease or condition being targeted. For instance, sodium chloride should be avoided in the context of the treatment of hypertension and cardiac condition. Such treatments will comprise administering the ESM peptides of the invention or a composition comprising the ESM peptides of the invention to a subject in need thereof, i.e. subject with or at risk of said diseases or conditions.

In other embodiments, the ESM peptides of the invention and pharmaceutical compositions comprising the same may be provided for use in the treatment of wounds, i.e. to promote wound healing. Such treatments will comprise administering the ESM peptides of the invention or a composition comprising the ESM peptides of the invention to a subject in need thereof, i.e. subject with a wound. Administration may be systemic or by direct application to the wound, e.g. its surface, its interior or its immediate vicinity.

Wounds, a breach in the integrity of, or denudement of, a tissue, commonly the skin, at an inevitable occurrence in the lives of humans and other animals. Wounds may be caused surgically, by physical injury (e.g. mechanical injuries; thermal injuries, for instance those resulting from excessive heat or cold; electrical injuries, for instance those caused by contact with sources of electrical potential; and radiation damage caused, for example, by prolonged, extensive exposure to infrared, ultraviolet or ionizing radiations) or by a spontaneously forming lesion such as a skin ulcer (e.g. a venous, diabetic or pressure ulcer), an anal fissure, a mouth ulcer and acne vulgaris.

-27 -In the medical fields, wounds are typically defined as either acute or chronic. Acute wounds are wounds that proceed orderly through the three recognised stages of the healing process following haemostasis (i.e. the inflammatory stage, the proliferative stage and the remodelling phase) without a protracted fimecourse.

Chronic wounds are defined as those which fail to heal, e.g. following excessive skin loss such as through burns. Such wounds do not complete the ordered sequence of biochemical events of the healing process because the wound becomes stalled in one of the healing stages. Commonly, chronic wounds are stalled in the inflammatory phase. Chronic wounds are a major source of morbidity for patients. The ESM peptides of the invention and pharmaceutical compositions comprising the same may be used in the treatment of acute or chronic wounds.

"Treatment" when used in relation to the treatment of a medical disease or condition in accordance with the invention is used broadly herein to include any therapeutic effect, i.e. any beneficial effect on the disease or condition. Thus, not only included is eradication or elimination of the disease or condition, or cure of the subject of the disease or condition, but also an improvement in the disease or condition or overall well-being of the subject or a reduction in the severity of the disease or the risk of a detrimental outcome associated with the disease or condition for the subject. Thus included, for example, is an improvement in any symptom or sign of the disease or condition, or in any clinically accepted indicator of the disease or condition. Treatment thus includes both curative and palliative therapy, e.g. of a pre-existing or diagnosed disease or condition, i.e. a reactionary treatment.

In the context of wounds specifically, treatment includes an improvement in any clinically accepted indicator of wound healing, for example a decrease in wound size (depth and/or area), an acceleration of healing time, or a reduction in general discomfort or pain in the wound or surrounding tissue. Treatment further includes any delay in, or limitation, reduction or prevention of, an increase in the size of the wound or the development of a chronic or poorly healing wound from an acute wound.

"Prevention" when used in relation to the treatment of a medical disease or condition in accordance with the invention is used broadly herein to include any prophylactic or preventative effect. It thus includes delaying, limiting, reducing or -28 -preventing the disease or condition or the onset of the disease or condition, or one or more symptoms or indications thereof, or reducing the eventual severity thereof, for example relative to the condition or symptom or indication prior to the prophylactic treatment. Prophylaxis thus explicitly includes both absolute prevention of occurrence or development of the disease or condition, or symptom or indication thereof, and any delay in the onset or development of the condition or symptom or indication, or reduction or limitation of the development or progression or eventual severity of the condition or symptom or indication. In the context of wounds, prevention includes preventing an acute wound developing into a chronic wound.

The subject may be any human or non-human animal subject, but more particularly may be a human or non-human vertebrate, e.g. a non-human animal selected from mammals, birds, amphibians, fish and reptiles. Mammalian subjects are preferred. The non-human animal may be a livestock or a domestic animal or an animal of commercial value, including laboratory animals or an animal in a zoo or game park.

Representative non-human animals therefore include dogs, cats, rabbits, mice, guinea pigs, hamsters, horses, pigs, sheep, goats, cows, chickens, turkeys, guinea fowl, ducks, geese, parrots, budgerigars, pigeons, salmon, trout, tilapia, catfish, bream, barramundi, grouper, mullet, amberjack, croaker, rohu, goby, cod, haddock, sea bass and carp. Veterinary uses of the invention are thus covered. The subject may be viewed as a patient. Preferably the subject is a human.

In other embodiments the water soluble peptides of the invention and compositions comprising the same find utility in wide variety of further applications including, but not limited to, the cosmetic industry and the food industry. Such applications are well-known in the art and it will be appreciated that the composition of the present invention could readily be adapted and directed towards such uses.

Thus, in one embodiment there is provided a cosmetic composition comprising water soluble eggshell membrane peptides obtained or obtainable from the methods described above together with at least one physiologically acceptable carrier, diluent or excipient. This composition may also comprise other cosmetic agents.

The cosmetic compositions of the invention can be applied to any external surface a subject's body. It will be appreciated that the cosmetic composition of the present -29 -invention may be applied alone or in combination with another form of cosmetic treatment or treatments.

More specifically, the ESM peptides of the invention may be incorporated, optionally together with other cosmetic agents, with one or more conventional carriers, diluents and/or excipients, to produce conventional cosmetic preparations such as creams, gels, foams, ointments, suspensions, emulsions, aerosols (as a solid or in a liquid medium), sprays and powders. Such compositions include, soaps, shampoos, shower gels, deodorants, make-up, hair styling consumables (hairsprays, hair gel, hair wax), hair dyes, shaving foams, aftershave, perfumes, skin moisturiser, face masks, toothpaste and mouthwash.

The compositions may additionally include lubricating agents, binding agents, glidants (flow aids), wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, colouring agents, moisturising agents and the like. Additional cosmetically active agents may be included in the cosmetic compositions.

The cosmetic compositions of the invention will typically comprise 1% to 99%, 5% to 95%, 10% to 90% or 25% to 75% of the ESM peptides of the invention, allowance being made for other ingredients.

In another embodiment there is provided a nutritional composition comprising water soluble eggshell membrane peptides obtained or obtainable from the methods described above together with at least one food stuff Suitable foodstuffs include but are not limited to dairy products (e.g. milk, yoghurt, cream, cheese, ice cream), fruit products (e.g. juice, purees, pulps, stews, preserves, sauces) vegetable products (e.g. juice, purees, pulps, stews, preserves, sauces, soups), bakery products (e.g. bread, bakes, biscuits) and meat products.

The peptides of the invention may be included in food products to provide a source of dietary protein or to functionally enhance the food, e.g. to improve digestive health or improve the condition of skin, hair and/or nails. The foodstuff may be a human foodstuff or a non-human animal foodstuff.

The invention also provides a feed additive or dietary supplement comprising water soluble eggshell membrane peptides obtained or obtainable from the methods described above together with at least one edible carrier, diluent or excipient.

-30 -The feed additive/dietary supplement may be provided in any convenient liquid or solid form, e.g. solutions, powders or granules, for adding to foodstuffs. Suitable diluents or excipients include binding agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, colouring agents, and the like. Examples include water, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, tragacanth, gelatine, calcium silicate, microcrystalline cellulose, cellulose, water syrup, water, water/ethanol, water/ glycol, water/polyethylene, hypertonic salt water, glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof.

The invention also provides a cell culture product comprising water soluble eggshell membrane peptides obtained or obtainable from the methods described above.

The peptides of the invention may be included in cell culture products to provide a source of protein for the cells being cultured or to functionally enhance the product, e.g. to improve stimulate growth, stimulate differentiation, promote survival or induce adherence to a solid surface. The cell culture product may be a culture medium comprising the peptides of the invention. The medium may be solid, semi solid or liquid, e.g. bacterial agar plates and bacterial broth systems (Mueller-Hinton, Peptone Water, MacConkey, Tryptic Soy) Dulbecco's Modified Eagle Medium, Eagle's Minimum Essential Medium, Roswell Park Memorial Institute (RPMI) 1640 Medium, Iscove's Modified Dulbecco's Media. In other embodiments the peptides of the invention are provided as a supplement for such media, such supplements may be the sterile solutions or powders prepared according to the methods described herein. In other embodiments the cell culture product may be a piece of disposable cell cultureware (consumables), e.g. formed from plastic, glass, silicone and/or metal, carrying the peptides on a surface which comes into contact with cells and/or medium when in use. Such items may be flasks, tubes, wells, plates, slides, vials and bottles.

The invention will be further described with reference to the following non-limiting

Examples, in which:

-31 -Figure 1 shows a flowchart with an overview of certain preferred embodiments of the method of the invention. In these embodiments eggshell membrane (ESM) flake or powder (101) is combined with 1 M sodium hydroxide solution (pH 14) (102) and hydrolysis takes place during incubation of the mixture at 120-125°C for 30 minutes at approx. 215kPa (103). The hydrolysis product is filtered to remove insoluble residues (104) and the pH of the filtrate is adjusted to pH 7.6 (105) with 0.05 to 0.1M citric acid (106). The neutralised peptide solution is diluted to a conductivity to 10-12 ms/cm (107) with purified water (108). The peptide solution is then sterile filtered with a 0.2pm cut off membrane (109) to provide a final soluble ESM peptide solution (110), which may be dried (111) to provide a soluble ESM peptide powder (112).

Figure 2 shows a UV spectral scan (200 -700 nm) of the peptide solution obtained in Example 1 and indicating absorbance at wavelengths corresponding to soluble peptides.

Figure 3 shows an SOS-PAGE gel on which the peptide solution obtained in Example 1 has been separated and indicating that a range of peptide sizes is obtained Figure 4 shows two images of the experiments performed in Example 3, wherein Fig. 4A shows wet ESM flake pulp combined with 1M sodium hydroxide (Bottle A), 30% w/v ESM dry flake in 1M ammonium hydroxide (Bottle B), and 30% w/v ESM dry flake in 1M sodium hydroxide control (Bottle C) prior to the heat treatment step; and Fig. 4B shows the wet ESM flake pulp combined with 1M sodium hydroxide (Bottle A), the 30% w/v ESM dry flake in 1M ammonium hydroxide (Bottle B), and the 30% w/v ESM dry flake in 1M sodium hydroxide control (Bottle C) after heat treatment.

-32 -

EXAMPLES

Example 1 -Hydrolysis of dry eggshell membrane powder and dry eggshell membrane flake with 1 M sodium hydroxide Hydrolysis of purified eggshell membrane powder (PEP) at high pH, pressure and temperature was attempted. It was found that 1M sodium hydroxide could be used to hydrolyse insoluble PEP into soluble peptides that could be filtered using a 0.2pm filter membrane (sterile filtration).

250g PEP (mean particle diameter 12 pm; >99% of the particles having a diameter less than 40 pm, prepared in accordance with WO 2016/066718) was added to 1 litre of 1M sodium hydroxide (pH 14) to form a 25% w/v mixture. The mixture was incubated for 30 minutes at 121 °C at a pressure of approximately 215 kPa. The reaction mixture was then filtered with a Propur SG 0.2 pm cartridge filter at room temperature. Filtrate was collected. The resultant solution had an ammonia smell due to the hydrolysis of the peptide bonds but there was no smell of hydrogen sulphide which is evidence that the disulphide bridges remained intact. Filtrate was adjusted to pH 7.6 with 0.1M citric acid at room temperature.

The resulting peptide solution was diluted further with purified water (1:50) to a conductivity 10-12ms/cm and was tested by spectrophotometry to confirm the presence of solubilised ESM peptides. Typically soluble proteins are detected and can be quantified at wavelengths of 214 and 280nm, the spectral scan shown in Figure 2 (200-700nm) measured significant peaks at these wavelengths for the diluted sample. Numerical readings from this scan are provided in Table 1 below.

Table 1

Wavelength (nm) Absorbance 280.0 1.204 278.0 1.184 242.0 3.953 -33 - 234.0 4.000 232.0 4.000 229.0 3.732 216.0 1.363 213.0 1.406 210.0 1.334 206.0 1.305 201.0 1.275 The presence and size characteristics of the soluble ESM peptides obtained was further demonstrated by using SDS-PAGE (Polyacrylamide Gel Electrophoresis).

Figure 3 shows a photograph of a 15% SDS-PAGE gel on which increasing amounts of the resulting peptide solution were separated. Lanes on which less than 10 pg of the resulting peptide solution have been separated show a distribution of peptide sizes, with particular bands at 17 and 10 kDa, which would represent peptides comprising 100 to 150 amino acids.

The diluted peptide solution was sterile filtered via a Corning 0.2 pm membrane bottle top filter into a sterile bottle. The sterilised peptide solution was transferred in 10m1 volumes into sterile bottles that were sealed with a sterile butyl stopper and crimp sealed. The sealed bottles remained stable and sterile during room temperature storage.

It was also demonstrated that the peptides in the hydrolysate retained intact disulphide bridges solution by adding 1M hydrochloric acid to the neutral ESM peptide solution and detecting a hydrogen sulphide smell. Without the further addition of acid the disulphide bridges in the peptides of the hydrolysate remain intact and at room temperature, the filtered hydrolysate is stable undergoing no further hydrolytic breakdown.

The soluble peptide liquid was then dried by lyophilisation and the resulting solid was reduced to a fine powder by milling. No problems such as caking were experienced. The resultant powder was shown to be 100% soluble in water and other aqueous buffers which means that it can easily be used to supplement existing formulations including a commercial cosmetic vitamin crème. Whilst the soluble peptide powder was found not to be directly soluble in 100% organic liquids -34 -it can be incorporated into organic formulations by first dissolving in a small amount of water which is then incorporated into the formulation.

The experiment was repeated using dry ESM flake in place of PEP. The ESM flake was prepared as described in WO 2015/058790 and then dried. The size of the dried flake is much greater than the powder. It was possible to solubilise 15% w/v of the dry ESM flake by the same method (data not shown).

Example 2 -Hydrolysis of purified eggshell membrane with 1 M ammonium hydroxide The experiment described in Example 1 was repeated using a weak base, 1M ammonium hydroxide, for the hydrolysis step. Using the same time, pressure and temperature for the hydrolysis reaction, it was found that there was no hydrolysis of ESM by the weak base at pH 11.5 (data not shown).

Example 3 -Hydrolysis of wet ESM flake pulp with 1 M NaOH The preparation of soluble peptides from ESM was also attempted using a wetted ESM flake pulp obtained as described in WO 2015/058790 as the substrate starting material for the alkaline hydrolysis step. This is the precursor of the dry ESM flake used in Example 1. The wetted pulp is obtainable from the final stage of ESM isolation process before the ESM is dried to a flake or powder form Wet ESM flake pulp was obtained as described in WO 2015/058790. The wet flake pulp consisted of approximately 20% solid ESM material holding 80% liquid. 780 g of the wet pulp was placed into a 2 litre bottle to which 600 ml of 1M sodium hydroxide (strong base) was added. This is approximately equivalent to a 15% w/v mixture of ESM flake and water. Calculated pH was approximately 13.5. At this stage, the wet pulp is not be completely submerged in the alkaline solution (Figure 4A). The mixture was then heat-treated at 121 °C for 30 minutes at a pressure of approximately 215 kPa using an autoclave wet cycle. After heat-treatment, only a dark solution remains with no wet pulp starting material being visible. It was -35 -observed that the pulp hydrolysate in solution had a final volume greater than 600 ml, which was the result of the volume contribution of the water held in the wet pulp.

As a positive control experiment, 30% w/v dried ESM flake starting material was hydrolysed using 1M sodium hydroxide as a strong base under the same physical conditions. The 30% w/v dry flake starting material was also hydrolysed.

As a further comparison, 30% w/v dried ESM flake starting material was treated with 1M ammonium hydroxide (pH 12.10) under the same physical conditions. The flake starting material remained unhydrolysed.

Figure 4 shows the results of each of the above-mentioned experiments. Specifically, Figure 4A shows, from left to right, the wet ESM flake pulp in 1M sodium hydroxide, approximately equivalent to 15% w/v ESM/water (Bottle A), the 30% w/v dry ESM flake in 1M ammonium hydroxide (Bottle B), and the 30% w/v dry ESM flake in 1M sodium hydroxide control (Bottle C) prior to the heat treatment step. Figure 4B shows, again from left to right, the wet pulp in 1M sodium hydroxide (Bottle A), the 30% w/v dry ESM flake in 1M ammonium hydroxide (Bottle B), and the 30% w/v dry ESM flake in 1M sodium hydroxide control (Bottle C) after the heat treatment step. It is clear from these images that 1M sodium hydroxide solution was able to hydrolyse both the wet pulp and dry flake ESM starting material and form an aqueous solution of soluble ESM. After heat treatment, Bottle C (i.e. dry ESM flake with 1M ammonium hydroxide) was unable to form an aqueous solution of ESM components.

It can be concluded that, even though the physical incubation conditions of hydrolysis were the same, the pH of the 1M ammonium hydroxide solution was too low to hydrolyse the ESM starting material.

Example 4-Pharmaceutical composition for wound treatment An example of a gel composition for administering water soluble ESM peptides obtainable in accordance with the invention directly to a wound is prepared with the following ingredients.

-36 -ESM peptides 1% Hydroxyethylcellulose 2.9% Sodium chloride 0.9% Calcium chloride 0.1% Tris Buffer Solution 10mM (pH 7.5) 95.1 Example 5 -Cosmetic composition An example of a topical cosmetic composition (a moisturising skincare body lotion) comprising water soluble ESM peptides obtainable in accordance with the invention is prepared with the following ingredients. This composition could also have therapeutic uses.

Oil phase: Mineral oil 3% Cyclomethicone 4% Isopropyl myristate 3% Stearic acid 1.8% Cetyl alcohol 1.0% Glyceryl stearate 1.5% Water phase: Carbomer 984 0.10% Glycerine 3% Thriethanolamine 0.90% ESM peptides 10% Water 71.7%

Claims

-37 -Claims 1. A method for preparing water soluble eggshell membrane (ESM) peptides, wherein said method comprises: (a) providing an at least 20% w/v mixture of water insoluble eggshell membrane and an aqueous solution of a strong base, wherein said mixture has a pH of at least 13.0 and is essentially free of eggshell (b) incubating said mixture at a temperature of about 110 °C to about 130°C and at a pressure of at least 150kPa for about 10 to about 30 minutes so as to form an aqueous solution of water soluble eggshell membrane peptides; (c) separating insoluble residues from the peptide solution formed in step (a); and (d) neutralising the residue-depleted peptide solution; and optionally (e) diluting the neutralised residue-depleted peptide solution with an aqueous liquid to a physiological conductivity, optionally wherein the neutralised residue-depleted peptide solution or the diluted neutralised residue-depleted peptide solution is filtered to remove cellular microorganisms 2. The method of claim 1, wherein the ESM is from chicken, duck, goose, turkey, guineafowl, quail, or ostrich.3. The method of claim 1 or claim 2, wherein the water insoluble ESM is in the form of a sheet, flake, shred or powder.4. The method of any one of claims 1 to 3, wherein the water insoluble ESM has a protein content of at least 80% w/w.5. The method of any one of claims 1 to 4, wherein the water insoluble ESM has a calcium carbonate content of no more than 2% w/w.-38 - 6. The method of any one of claims 1 to 5, wherein the pH of mixture of water insoluble eggshell membrane and an aqueous solution of a strong base is at least 13.5.7. The method of any one of claims 1 to 6, wherein the strong base is sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, strontium hydroxide, or combinations thereof 8. The method of any one of claims 1 to 7, wherein the aqueous mixture of a strong base and water insoluble ESM contains ESM at equal to or greater than 30% w/v.9. The method of any one of claims 1 to 8, wherein the incubation temperature is at least 120 C. 10. The method of any one of claims 1 to 9, wherein the incubation time is at least 20 minutes.11. The method of any one of claims 1 to 10, wherein the pressure is at least 200 kPa.12. The method of any one of claims 1 to 11, wherein the incubation time, pressure and temperature is about 121 t at about 215kPa for about 30 mins.13. The method of any one of claims 1 to 12, wherein the insoluble residues are separated from the peptide solution by filtration, preferably by filtration with a filter with a cut-off size of 0.45 pm or less.14. The method of any one of claims 1 to 13, wherein the residue-depleted solution is neutralised to a pH of about 7.0 or 7.6.-39 - 15. The method of any one of claims 1 to 14, wherein the residue-depleted solution is neutralised with hydrochloric acid or citric acid.16. The method of any one of claims 1 to 15, wherein the neutralised peptide solution is diluted to a conductivity of to 10 to 12 ms/cm.17. The method of any one of claims 1 to 16, wherein said method further comprises a step in which the neutralised residue-depleted peptide solution, the diluted neutralised residue-depleted peptide solution or the sterile filtered forms thereof is dried, thereby forming a dry soluble eggshell membrane peptide composition.18. Water soluble ESM peptides obtained or obtainable from the method defined in any one of claims 1 to 17.19. A composition comprising the water soluble ESM peptides as claimed in claim 18.A pharmaceutical composition comprising the water soluble ESM peptides as claimed in claim 18 together with at least one pharmaceutically acceptable carrier, diluent or excipient.21. The water soluble ESM peptides as claimed in claim 18 or the pharmaceutical composition as claimed in claim 20 for use in the treatment or prevention of a disease or condition treatable or preventable with ESM peptides.22. A cosmetic composition comprising the water soluble ESM peptides as claimed in claim 18 together with at least one physiologically acceptable carrier, diluent or excipient.23. A nutritional composition comprising the water soluble ESM peptides as claimed in claim 18 together with at least one food stuff -40 - 24. A feed additive or dietary supplement comprising the water soluble ESM peptides as claimed in claim 18 together with at least one edible carrier, diluent or excipient.25. A cell culture product comprising the water soluble ESM peptides as claimed in claim 18.