USE OF A BIOASSAY TO VALIDATE EFFICACY

Field

The present invention relates to processes for the validation of the efficacy of compositions having biological activity, particularly such compositions derived from milk, to provide quality control in a commercial production process .

Background

Milk, and products generated from milk fractions are recognised to provide nutritional value. Milk is a complex mixture of molecules, including numerous polypeptides, lipids and fats, and carbohydrates. The polypeptide component, while dominated by casein, contains many other proteins having diverse functions, such as OC-lactalbumin, β- lactoglobulin, immunoglobulins and caseinoglycomacropeptide .

Isolated or enriched milk components have been used as dietary supplements, often because of the ease in which the components of milk are digested. Nutraceuticals which contain milk components, particularly whey protein, have been used where the recipient desires or is in need of readily digestible protein. Applications for such nutraceuticals include body building, where it is desirable to maximise protein available for the creation of new muscle, or for administration to people who have difficulties in digesting or absorbing food normally, or who need to gain weight.

Milk components and hydrolysed components of milk have also been used to deliver bioactive components in edible form to subjects, such as angiotensin converting enzyme inhibiting peptides, glucagon-like peptide 1 and lactoferrin, or to reduce the risk of type 1 diabetes mellitus.

Many currently available nutraceuticals carry claims about their efficacy which are not substantiated by clinical results. Accordingly such products may not be efficacious,

over placebo effect. This problem is particularly so for milk-derived nutraceuticals .

It is an aim of a preferred embodiment to provide a method of validating the efficacy of a nutraceutical and confirming that a nutraceutical has the activity it purports to have .

Summary of Invention

In a first aspect the present invention provides a process for validating the efficacy of a sample having a known activity determined using a biological assay, the process comprising subjecting the sample to a biological assay capable of testing for the activity.

In a second aspect the present invention provides a process for validating the efficacy of a product, the process comprising

(a) determining a biological assay for a desired activity,

(b) testing a sample to validate its efficacy using a biological assay for the activity of (a) .

Steps (a) and (b) need not be carried out concurrently, or in the same site.

In a third aspect the invention provides a process for determining the ability of a nutraceutical composition to provide a biological activity of interest, comprising: subjecting at least a portion of a component of the nutraceutical composition to a biological assay capable of testing for the biological activity of interest; and determining the results of the biological assay, wherein a correlation between the results of the biological assay and the biological activity of interest indicates the ability of the nutraceutical composition to provide the biological activity of interest.

In a fourth aspect the invention provides a method of determining the ability of a nutraceutical composition to provide a biological activity of interest, the method comprising: subjecting a sample of a product that comprises the nutraceutical composition to a biological assay capable of detecting a biological activity associated with efficacy of the nutraceutical composition, wherein the detection of the biological activity associated with efficacy of the nutraceutical composition indicates that the nutraceutical composition is capable of providing a biological activity of interest compared to a placebo controlled composition.

In an embodiment of the fourth aspect the method further comprises obtaining a sample from an end product of the production process for the nutraceutical composition or an intermediate product formed during the process of producing the nutraceutical composition.

In an embodiment of the fourth aspect the sample of the product that comprises the nutraceutical composition is derived from a milk product.

In a further embodiment of the fourth aspect the sample of the product that comprises the nutraceutical composition is a milk fraction, such as a whey fraction such as whey protein isolate.

In a further embodiment the sample is a hydrolysed whey protein fraction comprising a complex mixture of proteins and peptides .

The method can be used to determine if a product has the ability to attenuate symptoms of muscle damage or enhance muscle contractile force by assessing the ability of a sample to induce cell proliferation in a cell proliferation assay for example using fibroblasts or myoblasts or to promote an anti-inflammatory response.

In their co-pending patent application AU2006903232 the inventors describe clinical trials which demonstrate that a composition comprising a hydrolysate of whey protein is able to attenuate a reduction in muscle function which results from muscle damage and/or is able to enhance recovery from muscle damage. This composition is a milk fraction, which was one of thousands of milk fractions tested by biological assays and shown to have a desirable biological activity. The production of this milk fraction has been scaled-up to produce the composition on a commercial scale. The inventors found that some of the product they were producing on a commercial scale did not have the biological activity that was expected. As the scale up process may not always produce products capable of having the desired biological activity, due to for example, faulty enzyme batch, overheating, etc. the inventors decided that a validation or quality control step should be included in the production process. This validation step involves testing a sample of the product for the desired biological activity. This allows the produces of the product to assure customers that they are purchasing a quality product which has the activity it purports to have.

This is different from prior art validation processes, particularly those of the pharmaceutical and food industry, where generally the composition of the sample is tested, to determine if there are contaminants. Here it is the activity of the sample that is tested to see if the sample is what is expected.

It is also different from research validation, where the proposed activity of a compound is tested by a biological assay to confirm that it has the activity that is predicted. In the present invention the process provides for quality control, by using the same biological assays used to determine the utility of a compound to check that a sample of the compound will actually have the activity expected.

The invention is particularly distinguished from the prior art as milk fractions, particularly whey fractions are a complex mixture of proteins, peptides and other components. It is not possible to easily elucidate if the actual composition of a fraction is as expected by comparing the levels of all components of the fraction. The present invention allows the fraction to be studied functionally, wherein if a fraction has a biological activity which correlates to a predetermined biological activity then that fraction passes quality control.

Brief Description of the Figures.

In the following examples, reference will be made to the accompanying figures as follows:

Figure 1 provides in graphical form the results of in vitro testing of test samples for their ability to influence fibroblast cell proliferation. "FCS" represents fetal calf serum. The test sample is a WPI hydrolysate and is indicated as WPI-H.

Figure 2 provides in graphical form the results of in vitro testing of test samples for their ability to influence the expression of the pro-inflammatory cytokines in a LPS stimulated macrophage cell line (TNFα (Fig 2A) and IL- lβ (Fig 2B) . The test sample is a WPI hydrolysate and is indicated as WPI-H.

Detailed Description

The present inventors have examined the biological activities provided by various orally-administered milk fractions .

The inventors have identified that Whey Protein Isolate (WPI), a milk fraction which contains whey proteins and which may be obtained from a variety of sources, including cheese whey and acid/casein whey, possesses an anti-inflammatory

activity as identified by an in vitro assay of TNFα expression inhibition. They have also identified that the hydrolysis of WPI unexpectedly results in an increase in this anti-inflammatory activity. Hydrolysis of whey protein also enhanced the ability of whey protein to stimulate the growth of fibroblasts in vitro.

The inventors have further identified that the administration of the enzyme hydrolysate of WPI is able to attenuate symptoms of muscle damage and promote the recovery of muscle function in subjects after muscle-damaging exercise. This activity is greater than the activity possessed by the corresponding non-hydrolysed form of WPI. The hydrolysate of WPI was also able to enhance the muscle force generating ability in subjects subjected to muscle- damaging exercise.

According to a preferred embodiment of the first and second aspects the biological assay used to determine the activity of a composition is also used to validate its efficacy.

Validating or validation as used herein refers to the confirmation of a property known to be possessed by the product of the process and therefore predicted to be possessed by the sample.

Efficacy as used herein refers to the ability of the sample to provide the desired amount of a desired effect.

Quality control as used herein refers to processes to verify that the product has the desired activity and therefore is of an expected quality.

A biological assay includes any cellular assay as herein before described.

Term biological activity as used herein refers to any activity of the sample on cells or tissues.

The term "sample" as used herein refers to a composition taken from a step in a commercial process. The sample may be

taken from an end product of the production process, or may be an intermediate in the process. The sample may also be a stored product, wherein the sample is tested to see if the product has maintained activity. The validation process may be used to determine the shelf life of the product, the effect of temperature and further processing steps, for example lyophilisation, encapsulation, etc.

The invention is particularly applicable to a sample comprising a complex mixture of components such as proteins and/or peptides. It is particularly envisaged that the invention be used for quality control of a commercial fractionation process, particularly a milk fractionation process .

By "end product" it is meant that the product is the end result of that process but does not mean that the end product cannot then go on to be used in other processes. For example a commercial process for producing a whey protein isolate hydrolysate may comprise the steps of enzyme hydrolysis and subsequent size fractionation. The product of the enzyme hydrolysis step is intended to be included in the scope of an "end product" even though this is then subjected to a further processing step (size fractionation). However in a preferred embodiment the end product is the final product that is collected from a commercial process for ultimate use or sale.

The invention is particularly applicable to validating the efficacy of nutraceuticals , particularly those derived from milk. However, the invention may be extrapolated to any product whose activity is determined using a cellular assay and whose production is not 100% guaranteed, giving the possibility that a sample of the product may not have the desired activity. This may be particularly the case if the product is produced by an enzyme or under stringent conditions .

Milk and its fractions have been proposed to have many- activities, as tested by biological assays. For example, a milk fraction was described in US2007110818 as having COX-2 inhibitory activity as tested by assaying for inhibition of prostaglandin E2 production from HUV-EC-C cells (a permanent endothelial cell line derived from the vein of a normal human umbilical cord; ATCC CRLl730; M. Miralpeix, M. Camacho et al., Brit. J. Pharmacol. 121 (1997), 171-180). The inventors' earlier application, Australian patent application 200690303232 describes milk fractions that improve muscle function or recovery as tested by assaying for TNFα inhibition or stimulation of fibroblast cell division in vitro. Another of the inventors' earlier applications, published as WO 2007/028211 describes that WGFE decreases post-exercise inflammatory responses in muscle as tested by assaying for reduced TNFα expression in RAW cells. Another of the inventors' earlier applications, published as WO 2007/028210 describes that WGFE and WPI increase muscle strength as tested by assaying for increased myoblast cell growth and fibroblast proliferation in vitro.

Preferable the biological assays are cell based assays. Other biological assays may include differential gene expression and biomarker analysis, motility, chemotaxis, contraction, relaxation, biosynthesis, secretion of signalling molecules, depolarization, repolarization, degranulation, adhesion, aggregation, change in metabolic rate, and immediate cellular responses and other assays known in the art .

Other biological activities for protein fractions from milk or other sources or whole protein extracts will be known to those skilled in the art, as will assays for evaluating such biological function.

In particular embodiments, the sample is a milk fraction. Throughout the specification the term "milk

fraction" refers to a composition which is ultimately derived from milk and which is at least enriched in one or more constituents found in whole milk. A "milk fraction" may be one which contains non-casein proteins found in milk. Whey or "milk plasma" is a common milk fraction which remains after the process of "curdling" has removed much of the casein and milk fat component from whole milk or skim milk. "Milk fraction" includes fractions which have been enriched for specific components or combinations of components of milk. The milk fraction "whey protein isolate" (WPI) for instance is one in which the non-casein protein components of milk have been enriched when compared with whole milk.

"Whey protein" comprises one or more protein (s) which are found in whey and which, when hydrolysed, possesses the desired biological activity described herein. Different sources of whey protein are contemplated. In particular, whey proteins originating from sweet whey or from acid whey have both been demonstrated to possess similar activity.

There are, for example, numerous methods for the production of whey, for instance as by-products arising during the production of different forms of cheese or as a by-product of the casein making process, and each of these will produce whey with a somewhat different composition. The principal proteins found in whey are α-lactalbumin and β- lactoglobulin and caseinoglycomacropeptide (CGMP) , the latter forming about 25% of protein present in cheese whey WPI. Milk fractions which contain one or more of these proteins are also contemplated.

The milk may be from cows, or due to the relatively similar composition of milk possessed by domestic species it is anticipated that milk from other animals including sheep, goats, horses and buffalo will be suitable. There may also be advantages in using whey protein of human origin, for

instance in the preparation of compositions for administration to newborn babies.

The methods of the present invention are particularly- useful for validating the efficacy of nutraceuticals .

A "nutraceutical" as defined herein represents an edible product isolated or purified from food, which is demonstrated to have a physiological benefit or to provide protection or attenuation of an acute or chronic disease or injury when orally administered. The nutraceutical may thus be presented in the form of a dietary supplement, either alone or admixed with edible foods or drinks.

The nutraceutical composition may be in any suitable form. For example, the nutritional composition may be in the form of a soluble powder, a liquid or a ready-to-drink formulation. Alternatively, the nutritional composition may be in solid form; for example in the form of a ready-to-eat bar or breakfast cereal, as a powder, or capsules or other form. Various flavours, fibres, sweeteners, and other additives may also be present.

The present invention is particularly suitable for quality control of commercial processes for fractionation of a product to give at least one biologically active fraction.

The product that is subjected to commercial fractionation is preferably milk or a milk fraction such as whey. The biologically active fraction may be prepared on a commercial scale by any means, for example by hydrolysis of the starting product, by size, charge or affinity separation or a combination of a plurality of fractionation methods.

Any of the steps used in commercial fractionation may lead to a loss in biological activity of the end product. Accordingly the present invention provides a method of quality control to check that a commercial process produces products that have the desired biological activity as determined by biological assays for that activity.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

As used in the present specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to a whey protein includes one or more whey proteins.

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

The present invention will now be described with reference to the following, non limiting examples:

Example 1. Preparation of Whey Protein Isolate

The aim of the WPI process is to enrich whey proteins by removing other whey components, such as water, lactose, fat, and ash (which comprises the mineral component of milk such as phosphorous, sodium, potassium, calcium, magnesium, and other metals) , until the remaining material is greater than 90% whey proteins (sample basis) . Processes which are commonly used commercially to produce WPI comprises steps of anion exchange chromatographic separation, ultrafiltration (UF) and spray drying.

The WPI production process works on the principle of anion exchange chromatography, with a large diameter resin which allows for a high flow rates with viscous materials. The starting material may be sweet whey produced by rennet coagulation or acid whey which results from the removal of casein by adding an acid. It is anticipated that whey produced by other methods, such as ultracentrifugation,

microfiltration or ethanol precipitation would also be suitable. Ideally the starting material for WPI production is WPC35 (whey protein concentrate 35% w protein/w solids) , which is a generic product produced by the ultrafiltration of raw whey to remove ash and lactose. This process is widely known to those in the dairy industry. A very similar product is also produced by using skim milk instead of whey.

The starting material is introduced onto anion exchange columns (GibcoCel CR201) to load the resin with predominantly negatively charged (at the pH of whey of 6.5) whey components. The columns are rinsed with water to remove unbound materials, and the bound whey components are eluted and the columns regenerated with a mixture containing 0.75 M sodium chloride and 0.75 M potassium chloride, after which the columns are rinsed to remove residual chloride.

The whey components eluted from the CSEP are desalted and concentrated by diafiltration through low temperature, low molecular weight, spiral ultrafiltration membranes. This step retains proteins, but removes salt, ash components and lactose. During this step total solids rise from 4% to 25% and the protein concentration rises from 50% of total solids to greater than 90% of total solids.

The purpose of the dryer is to remove the majority of the remaining water in the product until a maximum of 5% water remains. The dryer does this by atomizing the WPI concentrate in a chamber filled with hot air. As the resultant WPI powder is subsequently reconstituted in water as it is prepared for the hydrolysis, it may be possible to eliminate the drying step from this process.

The inventors have examined hydrolysates of WPI produced from cheese whey and from acid whey. Both contain similar bioactivity on enzyme digestion, and so it is anticipated that the type of WPI will not materially alter the resultant biological activity produced by the WPI hydrolysate.

WPI from each of sweet whey and acid whey WPI were used for the manufacture of the hydrolysates tested in vitro, but only sweet whey WPI hydrolysates were tested in vivo. The hydrolysates produced from sweet whey WPI and from acid whey WPI demonstrated very little difference in the in vitro assays, and therefore it is expected that the results for sweet whey WPI hydrolysates in vivo may be extended to acid whey hydrolysates .

Example 2. Hydrolysis of WPI to make WPI-H

The protease solution used in this example is a commercial product "Neutrase"® (Novozymes).

The target pH for the hydrolysis reaction was pH 6.5. The pH change during hydrolysis was limited, so it was practical to commence hydrolysis at pH 6.6 and not readjust the pH during the reaction. The final pH was around 6.4.

Three kilograms of WPI as prepared according to Example 1 was reconstituted in 27 1 water to make up 30 1 10 % (w/v) solids solution. The pH was adjusted to 6.6 with 4M NaOH prior to heating to 50 -C.

Once heating had commenced, the solution was stirred continuously.

4.5g of Neutrase ® 1.5MG (Novozyme) was dissolved in 45 ml water to prepare a 10 % solution, which was then added to the WPI solution. The pH was continually monitored and adjusted only if pH fell below 6.4. The hydrolysis process can be monitored in real time by the decrease in pH. The actual amount of hydrolysis that has taken place can be determined afterwards by measuring the "degree of hydrolysis" using a method based on o-phthalaldehyde (for example Lee et al . , 1978; supra) . The actual amount of hydrolysis taking place under the conditions described above is not very high, with an observed net degree of hydrolysis (%) between 0.3 and 3.0.

After 60 min hydrolysis the solution was adjusted to pH 4.0 with 4M HCl and the temperature maintained at 50 ² C for 30 min to deactivate the Neutrase ®. The resulting hydrolysate was cooled to 25 ^a C and the pH readjusted >pH 6.5, and ideally to pH 7.0.

The hydrolysate was then dried, ideally by freeze-drying at 35 ^S C.

Hydrolysates of WPI or other milk fractions which contain whey protein may be produced using other proteinase enzymes .

Example 3. Assaying for biological activity of WPI-H compared to WPI

The hydrolysate prepared according to the process described in example 2 was screened for activity in assays to determine inhibition of the expression of TNFα in macrophages or stimulation of fibroblast cell growth in vitro .

1) Cell Proliferation Assays:

Two cells lines were used for this analysis, a murine muscle myoblast cell line (C2C12; ATCC Number CRL-1772) and a murine fibroblast cell line (NIH/3T3, ATCC Number CRL-1658). Both these cell lines are adherent cells and are maintained in DMEM containing 10% serum (FBS for C2C12 ; new born calf serum for NIH/3T3) cultured at 37°C, 5% CO ₂ and split when the cells reach 80% confluency at a 1:5 - 1:10 ratio.

Cells were seeded into 96-well plates at a starting density of IxIO ⁴ cells/well in Dulbecco's modified Eagle's medium (DMEM) containing 10% serum (FBS for C2C12; NBS for NIH/3T3). The cells were cultured overnight at 37°C; 5% CO ₂ . The next day the serum containing media was removed and the cells washed in PBS. The cells were then cultured in 100 μl

serum free DMEM supplemented with test agents at a concentration of 2mg/ml (n=7) for 48 hrs at 37°C; 5% CO ₂ . To quantitate the cell growth 10 μl WST-I cell proliferation reagent (Roche) was added to each to each well and the cells incubated for a further 3 hours at 37 ⁰ C. During this time viable cells convert the WST-I reagent to a soluble formazan dye which was measured in a micro plate reader, the absorbance at 450 nm directly correlates to the cell number. Stimulation of cell growth by the agents was compared to a positive control (10% fetal calf serum) and un-hydrolysed WPI.

It can be seen from Figure 1 that the hydrolysate (WPI- H) showed greater than 15% more cell proliferation than WPI. This level of increased activity in this assay is used for quality control to validate that the process has produced a suitable hydrolysate.

2) Anti-Inflammatory Assays:

The human monocyte cell line THP-I (ATCC Number TIB-202) is used for this assay. This cell line grows as a cell suspension and is maintained in RPMI 1640 media supplemented with 10% FCS and 2 mM L-glutamine at 37 ⁰ C, 5% CO ₂ . The culture media is renewed every 2-3 days and then subcultured when the cell concentration reaches 8xlO ⁵ cells/ml. Cell density should never exceed IxIO ⁶ cells/ml.

a) Seeding and differentiation of cells into adherent macrophage - like cells

Cells were counted and resuspended at 1.5 x 10 ⁵ cells/ml in culture media containing 50 nM Phorbol myristate acetate (PMA) , cells were then seeded in to 96 well culture plates, lOOμl (1.5xlO ⁴ cells) /well. The cells were incubated at 37°C, 5% CO ₂ for 24 hours and at this point they have differentiated and are now adherent macrophage - like cells.

b) Induction of an inflammatory response

To induce an inflammatory response in the macrophage- like cells the media was removed and replaced with media containing 100 ng/ml LPS. To analyse the anti-inflammatory response replicate wells were supplemented with test agents at a concentration of 2mg/ml (n=7). The cultures weree then maintained at 37°C, 5% CO ₂ for 24 hours at which point the conditioned media was aspirated, replicate wells were pooled, then centrifuged at 15,000 g for 5 mins to remove cellular debris and stored at -20 ⁰ C.

c) Analysis of pro-Inflammatory cytokines.

An inflammatory response can be measured by the release of pro-Inflammatory cytokines into the conditioned media collected above; therefore an anti-inflammatory response will suppress the release of the cytokines. Two pro-inflammatory cytokines were measured - TNF α and IL-lβ, using the respective BD optEIA kits from BD Sciences as per manufacture's instructions.

Samples were analysed in quadruplicate. It can be seen from Figure 2 that the hydrolysate (WPI-H) showed greater than 15% less proinflammatory cytokine presence than WPI. The reduction in pro-inflammatory cytokines means that WPI-I has at least 15% greater anti-inflammatory activity than WPI. This level of increased activity in this assay is used for quality control to validate that the process has produced a suitable hydrolysate.

Example 4. Validation of Recovery Activity

Neutrase ® hydrolysed WPI was prepared as described in examples 1 and 2 and test samples validated using the assays described in Example 3. During the optimisation of process for the production of the hydrolysate, two thirds of the

samples tested for activity were found not to have the desired activity and were discarded. Now that that the process has been optimised 90% of the samples tested have the required efficacy of at least 15% increased activity compared to WPI.