WO2015085348A1

WO2015085348A1 - Apparatus for producing therapeutically active proteins in blood and uses thereof

Info

Publication number: WO2015085348A1
Application number: PCT/AU2014/001111
Authority: WO
Inventors: Jon FAIRWEATHER; David Connell
Original assignee: Cytokine Medical Australia Pty Ltd
Priority date: 2013-12-09
Filing date: 2014-12-09
Publication date: 2015-06-18
Also published as: AU2014361728A1

Abstract

The invention provides methods for producing therapeutically active proteins in a blood sample, the method comprising incubating a blood sample in a blood collection or storage device containing one or more substrates, the one or more substrates (a) having a density less than that of erythrocytes and greater than that of plasma and being capable of floating in the blood sample, (b) comprising silicates material or (c) comprising material capable of adhesion to one or more blood cells.

Description

APPARATUS FOR PRODUCING THERAPEUTICALLY ACTIVE PROTEINS IN BLOOD

AND USES THEREOF

FIELD

The present invention relates to apparatus for producing therapeutically active proteins in blood and methods for producing therapeutically active proteins using such apparatus.

BACKGROUND

US 6,713,246 and US 6,623,472, assigned to Orthogen Gentechnologic GmbH and Orthogen AG respectively, describe methods for enhancing the production of therapeutically effective proteins, such as interleukin-1 receptor antagonists (IL-1 Ra) in blood using a syringe made of glass, quartz or plastic, optionally filled with beads or granules to enlarge the internal surface area of the syringe (Orthogen 1 ) and/or utilising an inducer (Orthogen 2). Compared to recombinant technology, the Orthogen methods seek to provide a simple and inexpensive process for the production of proteins in blood by collecting blood from a patient and incubation of the blood sample to produce therapeutic proteins in the blood. Blood containing the therapeutic proteins can then be administered to a patient. In practice, the syringe sold commercially by Orthogen AG requires specialist apparatus to carry out the Orthogen methods. This means a clinic wishing to offer Orthogen's methods requires significant capital outlay to establish, which cost inevitably flows through to the cost to the patient.

It is an aim of an embodiment of the present invention to provide an alternative to the Orthogen methods which does not require specialist equipment.

It is a further aim of an embodiment of the present invention to provide a method for increasing cytokine production in blood on a commercial scale.

SUMMARY

A first aspect provides a method of producing a therapeutically active protein in a blood sample, the method comprising the step of incubating the blood sample in a blood collection or storage device containing one or more substrates, the one or more substrates having a density less than that of erythrocytes and greater than that of plasma or serum and being capable of floating in the blood sample.

Under the influence of gravity or centrifugal force, blood spontaneously sediments into three layers. At equilibrium the top, low-density layer is a straw-coloured clear fluid called plasma. Plasma is a water solution of salts, metabolites, peptides, and many proteins ranging from small (insulin) to very large (complement components). The bottom, high-density layer is a deep red viscous fluid comprising anuclear red blood cells (erythrocytes) specialized for oxygen transport. The relative volume of whole blood that consists of erythrocytes is called the hematocrit, and in normal human beings can range from about 38% to about 54%.

The intermediate layer is the smallest, appearing as a thin white band on top the erythrocyte layer and below the plasma, and is called the buffy coat. The buffy coat itself has two major components, nucleated leukocytes (white blood cells) and anuclear smaller bodies called platelets (or thrombocytes).

In the Orthogen methods, the granules or beads lie at the bottom of the syringe and thus are mainly in contact with the erythrocyte layer. The inventors proposed that it would be beneficial to have the erthyrocytes initially exposed to granules or beads (substrates) which are then located at the plasma/buffy coat interface upon separation of layers. Using substrates of a suitable density to float at this interface the inventors were able to produce more (weighted mean) of IL-1 Ra than the control Orthogen method 1 or substantially more of IL-1 Ra using a standard blood collection or storage vessel (not containing a substrate). The inventors also identified increased prolnterleukin 1-beta (IL-Ιβ), Interleukin 1 Receptor Antagonist (IL-1 Ra), Interleukin 2 (IL-2), Interleukin 4 (IL-4), Interleukin 5 (IL-5), Interleukin 6 (IL-6), Interleukin 7 (IL-7), Interleukin 8 (IL-8), Interleukin 9 (IL-9), Interleukin 10 (IL-10), Interleukin 12 (IL-12), Interleukin 13 (IL-13), Interleukin 15 (IL-15), Interleukin 17 (IL-17), Eotaxin 1 , Fibroblast Growth Factor-beta (FGFb), Vascular Endothelial Growth Factor (VEGF), Granulocyte Stimulating Growth Factor (G-CSF), Granulocyte Macrophage

Stimulating Growth Factor (GM-CSF), Platelet Derived Growth Factor (PDGF), Macrophage Inflammatory Protein 1a (MIP-1a), Macrophage Inflammatory Protein 1β (ΜΙΡ-1β),

Monocyte Chemoattractant Protein-1 (MCP-1 ), Interferon gamma (IFN-γ), Interferon gamma induced protein (IP-10), Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES), Tumour Necrosis Factor alpha (TNF-a) and immunoglobulins. The Orthogen method is only disclosed as producing IL-4, IL-10, soluble TNF-type I, soluble TNF-type II and IL-1 ra.

A second aspect provides an apparatus for producing a therapeutically active protein in a blood sample, the apparatus comprising a blood collection or blood storage device containing one or more substrates, the one or more substrates having a density less than that of erythrocytes and greater than that of plasma or serum and being capable of floating in the blood sample. ln one embodiment the method of the first aspect modulates the concentration of other proteins in the blood sample. The method may increase or decrease the concentration of one or more other proteins in the blood sample.

A third aspect provides a method for treating a subject in need thereof with a therapeutic protein, the method comprising incubating a blood sample in a blood collection or storage device containing one or more substrates, the one or more substrates having a density less than that of erythrocytes and greater than that of plasma or serum and being capable of floating in the blood sample, for a time sufficient to produce a therapeutic protein and administering the blood sample to the subject.

In one embodiment the subject has rheumatism, osteoarthritis and/or a back disorder or exhibits symptoms thereof.

In one embodiment of the first, second or third aspect the therapeutic protein is interleukin 1 receptor antagonist (IL-1 Ra), prolnterleukin 1-beta (IL-Ιβ), Interleukin 2 (IL-2), Interleukin 4 (IL-4), Interleukin 5 (IL-5), Interleukin 6 (IL-6), Interleukin 7 (IL-7), Interleukin 8 (IL-8), Interleukin 9 (IL-9), Interleukin 10 (IL-10), Interleukin 12 (IL-12), Interleukin 13 (IL-13), Interleukin 15 (IL-15), Interleukin 17 (IL-17), Eotaxin 1 , Fibroblast Growth Factor-beta (FGFb), Vascular Endothelial Growth Factor (VEGF), Granulocyte Stimulating Growth Factor (G-CSF), Granulocyte Macrophage Stimulating Growth Factor (GM-CSF), Platelet Derived Growth Factor (PDGF), Macrophage Inflammatory Protein 1a (MIP-1a), Macrophage Inflammatory Protein 1β (ΜΙΡ-1β), Monocyte Chemoattractant Protein-1 (MCP-1 ), Interferon gamma (IFN-γ), Interferon gamma induced protein (IP-10), Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES), Tumour Necrosis Factor alpha (TNF-a) and one or more immunoglobulins.

In one embodiment the substrate is capable of floating in the buffy coat of the blood sample.

In one embodiment the blood administered to the subject is their own blood or a fraction thereof (autologous treatment).

A fourth aspect provides a method for producing a therapeutically active protein in a blood sample, the method comprising incubating the blood sample in a blood collection or storage device containing one or more substrates, the one or more substrates comprising silicates material comprising minerals or other anionic species.

In one embodiment the silicates material is selected from one or more of silicon dioxide, silicic acid gel, precipitate silicon dioxide, silica gel, hydrous silica, hydrated silicic acid or poylsilicic acid gels of the general formula H₂n₊2Sin0₃n+i.mH₂0. In one embodiment the substrate is coated with an inert amorphous silicate material comprising one or more of phyllosilicates, kaolin, celite, bentonite, attapulgite and beidellite.

The inventors have found that use of substrates of silicates material provides more (weighted mean) of IL-1 Ra than the control Orthogen method 1 or substantially more of IL- 1 Ra using a standard blood collection or storage vessel (not containing a substrate). The inventors also identified increased prolnterleukin 1 -beta (IL-Ιβ), Interleukin 1 Receptor Antagonist (IL-1 Ra), Interleukin 2 (IL-2), Interleukin 4 (IL-4), Interleukin 5 (IL-5), Interleukin 6 (IL-6), Interleukin 7 (IL-7), Interleukin 8 (IL-8), Interleukin 9 (IL-9), Interleukin 10 (IL-10), Interleukin 12 (IL-12), Interleukin 13 (IL-13), Interleukin 15 (IL-15), Interleukin 17 (IL-17), Eotaxin 1 , Fibroblast Growth Factor-beta (FGFb), Vascular Endothelial Growth Factor (VEGF), Granulocyte Stimulating Growth Factor (G-CSF), Granulocyte Macrophage

Monocyte Chemoattractant Protein-1 (MCP-1 ), Interferon gamma (IFN-γ), Interferon gamma induced protein (IP-10), Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES), Tumour Necrosis Factor alpha (TNF-a) and one or more immunoglobulins. The Orthogen method is only disclosed as producing IL-4, IL-10, soluble TNF-type I, soluble TNF-type II and IL-1 ra.

The inventors propose that the silicates substrate increases the surface area in contact with and interacting with the blood cells.

A fifth aspect provides an apparatus for producing a therapeutically active protein in a blood sample, the apparatus comprising a blood collection or storage device containing one or more substrates comprising silicates material comprising minerals or other anionic species.

In one embodiment the silicates material is selected from one or more of silicon dioxide, silicic acid gel, precipitate silicon dioxide, silica gel, hydrous silica, hydrated silicic acid or poylsilicic acid gels of the general formula H₂n₊2Sin0₃n₊i.mH₂0.

In one embodiment the substrate is coated with an inert amorphous silicate material comprising one or more of phyllosilicates, kaolin, celite, bentonite, attapulgite and beidellite.

A sixth aspect provides a method for treating a subject in need thereof with a therapeutic protein, the method comprising incubating a blood sample in a blood collection or storage device containing one or more substrates comprising silicates material comprising minerals or other anionic species. ln one embodiment the silicates material is selected from one or more of silicon dioxide, silicic acid gel, precipitate silicon dioxide, silica gel, hydrous silica, hydrated silicic acid or poylsilicic acid gels of the general formula H₂n+2Sin03n₊i.mH₂0.

In one embodiment of the fourth, fifth or sixth aspect the therapeutic protein is interleukin 1 receptor antagonist (IL-1 Ra), prolnterleukin 1-beta (IL-Ιβ), Interleukin 2 (IL-2), Interleukin 4 (IL-4), Interleukin 5 (IL-5), Interleukin 6 (IL-6), Interleukin 7 (IL-7), Interleukin 8 (IL-8), Interleukin 9 (IL-9), Interleukin 10 (IL-10), Interleukin 12 (IL-12), Interleukin 13 (IL-13), Interleukin 15 (IL-15), Interleukin 17 (IL-17), Eotaxin 1 , Fibroblast Growth Factor-beta (FGFb), Vascular Endothelial Growth Factor (VEGF), Granulocyte Stimulating Growth Factor (G-CSF), Granulocyte Macrophage Stimulating Growth Factor (GM-CSF), Platelet Derived Growth Factor (PDGF), Macrophage Inflammatory Protein 1a (MIP-1a), Macrophage

Inflammatory Protein 1β (ΜΙΡ-1β), Monocyte Chemoattractant Protein-1 (MCP-1 ), Interferon gamma (IFN-γ), Interferon gamma induced protein (IP-10), Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES), Tumour Necrosis Factor alpha (TNF-a) and one or more immunoglobulins.

A seventh aspect provides an apparatus for producing a therapeutically active protein in a blood sample, the apparatus comprising a blood collection or storage device containing one or more substrates, the one or more substrates comprising material capable of adhesion to one or more blood cells.

The inventors have found that use of substrates capable of adhering to blood cells in the collection or storage device provides more (weighted mean) of IL-1 Ra than the control Orthogen method 1 or substantially more of IL-1 Ra than obtained using a standard blood collection or storage vessel (not containing a substrate). The inventors also identified increased prolnterleukin 1 -beta (IL-1 β ), Interleukin 1 Receptor Antagonist (IL-1 Ra),

Interleukin 2 (IL-2), Interleukin 4 (IL-4), Interleukin 5 (IL-5), Interleukin 6 (IL-6), Interleukin 7 (IL-7), Interleukin 8 (IL-8), Interleukin 9 (IL-9), Interleukin 10 (IL-10), Interleukin 12 (IL-12), Interleukin 13 (IL-13), Interleukin 15 (IL-15), Interleukin 17 (IL-17), Eotaxin 1 , Fibroblast Growth Factor-beta (FGFb), Vascular Endothelial Growth Factor (VEGF), Granulocyte Stimulating Growth Factor (G-CSF), Granulocyte Macrophage Stimulating Growth Factor (GM-CSF), Platelet Derived Growth Factor (PDGF), Macrophage Inflammatory Protein 1a (MIP-1a), Macrophage Inflammatory Protein 1β (ΜΙΡ-1β), Monocyte Chemoattractant Protein-1 (MCP-1 ), Interferon gamma (IFN-γ), Interferon gamma induced protein (IP-10), Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES), Tumour Necrosis Factor alpha (TNF-a) and immunoglobulins. The Orthogen method is only disclosed as producing IL-4, IL-10, soluble TNF-type I, soluble TNF-type II and IL-1 ra.

The inventors propose that the blood cells may secrete cytokines in response to adhesion to the substrate.

An eighth aspect provides a method of producing a therapeutically active protein in a blood sample, the method comprising the steps of incubating a blood sample in a blood collection or storage device containing one or more substrates comprising material capable of adhesion to one or more blood cells.

A ninth aspect provides a method for treating a subject in need thereof with a therapeutic protein, the method comprising incubating a blood sample in a blood collection or storage device containing one or more substrates comprising material capable of adhesion to one or more blood cells. In one embodiment the subject has rheumatism, osteoarthritis and/or a back disorder or exhibits symptoms thereof.

In one embodiment of the seventh, eighth or ninth aspect the therapeutic protein is interleukin 1 receptor antagonist (IL-1 Ra), prolnterleukin 1-beta (IL-Ιβ), Interleukin 2 (IL-2), Interleukin 4 (IL-4), Interleukin 5 (IL-5), Interleukin 6 (IL-6), Interleukin 7 (IL-7), Interleukin 8 (IL-8), Interleukin 9 (IL-9), Interleukin 10 (IL-10), Interleukin 12 (IL-12), Interleukin 13 (IL-13), Interleukin 15 (IL-15), Interleukin 17 (IL-17), Eotaxin 1 , Fibroblast Growth Factor-beta (FGFb), Vascular Endothelial Growth Factor (VEGF), Granulocyte Stimulating Growth Factor (G-CSF), Granulocyte Macrophage Stimulating Growth Factor (GM-CSF), Platelet Derived Growth Factor (PDGF), Macrophage Inflammatory Protein 1a (MIP-1a), Macrophage

Inflammatory Protein 1β (ΜΙΡ-1β), Monocyte Chemoattractant Protein-1 (MCP-1 ), Interferon gamma (IFN-γ), Interferon gamma induced protein (IP-10), Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES), Tumour Necrosis Factor alpha (TNF-a) and/or one or more immunoglobulins.

A tenth aspect provides a method of increasing the cytokine and immunoglobulin content of a blood sample, the method comprising incubating or storing the blood sample in the apparatus of any one of the second, fifth or seventh aspects. An eleventh aspect provides a treated blood sample from a subject, the treated blood sample comprising elevated levels of one or more therapeutically active proteins compared to a blood sample from the subject, in which the treated blood sample is provided by incubating or storing the blood sample in the apparatus of any one of the first, fourth or seventh aspects for such a time to elevate levels of one or more therapeutically active proteins in the sample.

An eleventh aspect provides a pharmaceutical or veterinary formulation comprising one or more therapeutic protein(s) produced by the first, fourth and eighth aspects. BRIEF DESCRIPTION OF THE DRAWINGS

In the figures that follow:

Figure 1 shows a diagrammatic representation of the apparatus of the second, fifth and seventh aspects.

Figure 2 a-d shows the biochemical analysis of blood treated according to the first, fourth and eighth aspects and blood treated according to Orthogen method 1

DETAILED DESCRIPTION

The invention allows the use of standard apparatus and need not require specialist machinery.

A blood collection or storage device is any apparatus suitable for collecting blood from a subject or storing such blood. In one embodiment it is a syringe. In another embodiment it is an evacuated container blood collection tube or similar, such as a vacutainer. In one embodiment it is a blood bag. In one embodiment it is tubing which connects to a blood bag or other blood storage device.

The blood collection or storage device may be made of glass, plastic, quartz, silicate or other suitable materials known in the art or a combination of such materials. In one embodiment the blood collection device is a S-Monovette Serum Blood Collection Tube from SARSTEDT AG & Co. (SarstedtstraBe, Postfach 1220, 51582 Numbrecht, Germany)

In one embodiment of all aspects the blood collection or storage device is sterile or is sterilised before use. .

The blood collection or storage device may be a 1.2-9.0 ml blood collection vessel (aspirate or evacuated container), or a blood collection bag (being comprised of PVC or non- PVC, and with volume 50 ml - 5000 ml), or glass container (vial or bottle), or culture vessel (polycarbonate, polyetylene terapthalate or polypropylene and with volume 15 ml - 1000 ml). The substrate(s) may take up 5% of the volume of the blood collection device. In other embodiments the substrate(s) may take up 2.5, 10, 20, or up to 50% of the volume of the blood collection device.

In one embodiment the substrate(s) provide(s) a column in the blood collection or storage device capable of allowing the blood flow therethrough.

The substrate(s) having a density less than that of erythrocytes and greater than that of plasma is/are intended to be positioned at the plasma/buffy coat interface when the blood collection device contains whole blood.

At room temperature and pressure the density of blood plasma is 1025 g/ml and the density of erythrocytes is around 1 125g/ml depending on the haematocrit levels. In one embodiment the substrate(s) have a density of between 1025 and 1 125 g/ml. The substrate may have a density of 1030, 1035, 1040, 1045, 1050, 1055, 1060, 1065, 1070, 1075, 1080, 1085, 1090, 1095, 1 100, 1 105, 1 1 10, 1 1 15 or 1 120 g/ml, with a variation in density of 1 ,2 or a 3 g/ml around each integer, when measured at 25°C and atmospheric pressure.

In one embodiment of the first, second or third aspects, the substrate is made of material, particularly polystyrene (approximate density 1030-1070g/ml), ABS (approximate density 1050g/ml),polyamide (approximate density 1 100 g/ml), polyethylene (approximate density 0.95-1.5 g/cc), poylpropylene (approximate density 0.9 g/cc), zirconium (approximate density 3.7 g/cc), stabilised zirconium formulations (approximate density 5.8-6.0 g/cc), cellulose acetate (approximate density ~1.3 g/cc), silica (approximate density 1.8 g/cc), poylmethylmethacrylate (approximate density 1.2 g/cc), cellulose acetate (approximate density 1 .3 g/cc) or silicates material (density 160- 960 g/ml) comprising comprising minerals or other anionic species. In one embodiment the silicates material is selected from one or more of silicon dioxide, silicic acid gel, precipitate silicon dioxide, silica gel, hydrous silica, hydrated silicic acid or poylsilicic acid gels of the general formula H₂n+2Sin03n₊i.mH₂0.

The substrate of the first, second or third aspect can be any shape or size provided it can be positioned at the plasma/buffy coat interface when the blood collection device contains whole blood. In one embodiment the substrate comprises granules, spheres, hemispheres, cubes, cuboids, pyramids, cylinders, tetrahedrons, octahedrons,

dodecahedrons, cones, needles, rods, prisms, balls or grains of material of the overall required density. In one embodiment the substrate comprises granules with a diameter of 0.5 to 5 mm.

The silicates material used in the substrate(s) of the fourth to sixth aspects or the material capable of adhesion to one or more blood cells used in the substrates of the seventh, eighth or ninth aspects may be coated onto the substrate such that it covers the entire or part of the surface of the substrate or form some or all of the substrate. In one embodiment the substrate comprises beads or granules of another material, e.g. glass, metal or plastics, completely or partly coated with one or more layers of the silicates material or material capable of adhesion to one or more blood cells. In one embodiment the substrate is entirely made of the silicates material or material capable of adhesion to one or more blood cells. In one embodiment the substrate comprises silicate granules.

In one embodiment the substrate(s) are irregular in shape, wherein the imperfections provide increased surface area and binding capacity.

The substrate(s) may be coated to selectively produce or block anti- or proinflammatory proteins.

The substrate(s) may be used in combination with an additive that selectively produces or blocks anti- or pro-inflammatory proteins.

In the methods of the first and fourth aspects blood in the apparatus is incubated to produce the therapeutically active protein(s) in the blood. In one embodiment incubation comprises holding the temperature of the blood sample between 20 and 40°C, preferably 37 °C over a period of 0.5 to 48 hours or more.

The therapeutic protein(s) produced by the methods of the first, fourth and eighth aspects can advantageously be modified, for example glycosylated.

In one embodiment the blood is incubated with an anticoagulant such as heparin, acid citrate dextrose solution, citrate phosphate dextrose solution with or without adening, sodium citrate or hirudin.

In one embodiment the blood enriched for the therapeutic protein can be

administered to a subject in need thereof without further manipulation or the need to transfer to another container. Alternatively the blood enriched for a therapeutic protein can be fractionated to remove solid components, particularly erythrocytes, using sterile filtration or centrifugation. In one embodiment, the proposed blood collection tubes are of a size able to fit in a standard centrifuge. In one embodiment the enriched plasma/serum can be stored (at <-19 °C in accordance with regulations in Australia, at higher temperature (2-8 °C) or even lower temperature (<-78 °C)) for later use.

In one embodiment the therapeutically active protein is a cytokine.

The term "cytokine" is a generic term for proteins released by one cell population which act on another cell population as intercellular mediators. Examples of such cytokines as produced from blood cells are Interleukin 1-beta (IL-Ιβ ), Interleukin 1 Receptor Antagonist (IL-1 Ra), Interleukin 2 (IL-2), Interleukin 4 (IL-4), Interleukin 5 (IL-5), Interleukin 6 (IL-6),

Interleukin 7 (IL-7), Interleukin 8 (IL-8), Interleukin 9 (IL-9), Interleukin 10 (IL-10), Interleukin 12 (IL-12), Interleukin 13 (IL-13), Interleukin 15 (IL-15), Interleukin 17 (IL-17), Eotaxin 1 , Fibroblast Growth Factor-beta (FGFb), Vascular Endothelial Growth Factor (VEGF), Granulocyte Stimulating Growth Factor (G-CSF), Granulocyte Macrophage Stimulating Growth Factor (GM-CSF), Platelet Derived Growth Factor (PDGF), Macrophage

Inflammatory Protein 1 a (MIP-1a), Macrophage Inflammatory Protein 1β (ΜΙΡ-1β),

Monocyte Chemoattractant Protein-1 (MCP-1 ), Interferon gamma (IFN-γ), Interferon gamma induced protein (IP-10), Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES), Tumour Necrosis Factor alpha (TNF-a) and/or one or more immunoglobulins.

Particularly the cytokine is a pro-inflammatory cytokine.

The subject treated by the methods of the third, sixth or ninth aspects may have rheumatoid arthritis or osteoarthritis and/or a back disorder or exhibits symptoms thereof.

The back disorder may be associated with neurological causation, such as sciatica. The blood or blood fraction enriched for the therapeutic protein may be administered to the subject by injection, for example, at a nerve root, into the joint or into the intervertebral disk.

Suitable treatments for such diseases previously involved an invertebral disk operation, cortisone treatment, or irrigation with saline or similar. While Orthogen's methods have been used to treat these conditions the present invention provides a more cost effective solution.

The subject is preferably an animal, In one embodiment the subject is classified as a mammal, including humans, rodents (e.g., mice and rats), and monkeys; domestic and farm animals; and zoo, sports, laboratory, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. In some embodiments, the mammal is selected from a human, rodent, or monkey. Similarly, "subject" for the purposes of treatment, refers to a mammalian subject, and includes both human and veterinary subjects.

Further advantageous configurations of the invention are evident from the dependent claims.

The invention will now be illustrated with reference to the following examples, which are not intended to be limiting. EXAMPLE 1

Blood Collection for Analysis.

Freshly drawn human blood was collected from each of either five or twenty (5 or 20, healthy, male and female in the age range 20-50 years, without anti-coagulants, anti- inflammatories or other illness, into three tubes individually containing (a) no granules (Neutral S-monovette 9 ml, Sarstedt AG& Co.), (b) chemically modified glass granules (Orthokine EOT II, 60 ml or 10 ml, Orthogen AG) and (c) beads coated with silicates (S- monovette Serum 9 ml, Sarstedt AG& Co.). These tubes were obtained pre-sterilised from the Manufacturers (specified).

The tubes were incubated under aseptic conditions at 37 °C for 24 h or different intervals, as indicated. After incubation, each container serum was centrifuged (3500 rpm, 10 min) and the upper layer, being plasma or serum, was transferred to a sterile syringe via a sterilising grade (0.22 urn) 33 mm filter disc membrane into a sterile syringe (3 ml, Terumo) and fitted with a sterile syringe cap (Combi Stopper, B Braun). The syringe was frozen (-21 °C ± -2 °C) for a period up to 7 days prior to analysis.

The levels of a suite of cytokines (including chemokines, interleukins and monokines) and growth factors in the plasma/serum were measured by Bio-Plex multiplex system (Bio- Rad, USA) with assays performed to the manufacturer's instructions. Data were graphed using Bio-Plex Data Pro and Microsoft Excel 2007.

The results are shown in Figure 2.

It can be seen from Figure 2a that the method of the invention produced more IL-6, IL-8, MIP-1 a and PDGF-bb than the Orthogen method.

It can be seen from Figure 2b that the method of the invention produced more G- CSF, MCP-1 , IL-1 ra, RNATES, TNF-a and IL-1 b than the Orthogen method.

It can be seen from Figure 2c that the method of the invention produced more

Eotaxin, IFN-g, MIP-1 b and VEGF than the Orthogen method.

It can be seen from Figure 2d that the method of the invention produced more IL-4, IL-7 and IL-13 than the Orthogen method. EXAMPLE 2

Blood Collection for Clinical Treatment.

Freshly drawn human blood was collected from patients into six (6) tubes, each containing beads coated with silicates (S-monovette Serum 9 ml, Sarstedt AG& Co.). These tubes are provided as pre-sterilised and collection ready by the Manufacturer (specified). The tubes were incubated under aseptic conditions at 37 °C for 24 h. After incubation, each tube was centrifuged (3500 rpm, 10 min) and the upper layer, being plasma or serum, was transferred to a sterile syringe via a sterilising grade (0.22 um) 33 mm filter disc membrane into a sterile syringe (3 ml, Terumo) and fitted with a sterile syringe cap (Combi Stopper, B Braun). The syringe was frozen (-21 °C ± -2 °C) for a period up to 6 weeks prior to clinical treatment.

Each patient was treated with weekly injections (1 injection per week for 6 weeks). The results were as follows:

Abbreviations:

VAS - visual analogue score for pain

KOOS - knee injury and osteoarthritis outcome score

MHH - modified Harris hip score

HOOS - hip dysfunction and osteoarthritis outcome score

FADI - foot and ankle disability index

ASES - American shoulder and elbow surgeons score

OXFORD - Oxford shoulder score

KNEE: Immediately After Therapy (39 patients)

Initial VAS (mean)

After VAS (mean)

Average reduction

Initial KOOS (mean) = 50.26

After KOOS (mean) = 62.32

Average increase = 12.06 VAS KOOS improve 30 76.92% 31 79.49% worse 8 20.51 % 7 17.95% no diff 1 2.56% 1 2.56%

KNEE: 3 month follow-up (46 patients)

Initial VAS (mean) = 5.78

After VAS (mean) = 4.24

Average reduction = 1.54

Initial KOOS (mean) = 50.66

After KOOS (mean) = 61.73

Average increase = 1 1.07

HIP: Immediately After Therapy (9 patients)

Initial VAS (mean)

After VAS (mean)

Average reduction

HIP: 3 month follow-up (1 1 patients)

· Initial VAS (mean) = 6.55

• After VAS (mean) = 4.82

• Average reduction = 1.73 Initial MHH (mean) = 47.82 After MHH (mean) = 51.82

Average increase = 4.00

Initial HOOS (mean) = 48.43

After HOOS (mean) = 57.28

Average increase = 8.85

ANKLE: Immediately After Therapy (4 patients)

• Initial VAS (mean) = 4.00

• After VAS (mean) = 2.00

• Average reduction = 2.00

• Initial FADI (mean) = 66.83

• After FADI (mean) = 75.23

• Average increase = 8.40

ANKLE: 3 month follow-up (5 patients)

• Initial VAS (mean) = 4.4

• After VAS (mean) = 3.6

• Average reduction = 0.8 · Initial FADI (mean) = 64.8

• After FADI (mean) = 69.1

• Average increase = 4.26 VAS FADI

improve 3 60.00% 3 60.00% worse 2 40.00% 2 40.00% no diff 0 0.00% 0 0.00%

SHOULDER Immediately After Therapy (4 patients)

Initial VAS (mean) = 5.00

After VAS (mean) = 4.75

Average reduction = 0.25

Initial ASES (mean) = 37.92

After ASES (mean) = 46.25

Average increase = 8.33

Initial HOOS (mean) = 24.75

After HOOS (mean) = 27.75

Average increase = 3.00

SHOULDER 3 month follow-up (7 patients)

• Initial VAS (mean) = 5.57

• After VAS (mean) = 4.00

• Average reduction = 1.57

• Initial ASES (mean) = 47.86

• After ASES (mean) = 62.13

• Average increase = 14.27 · Initial HOOS (mean) = 28.29

• After HOOS (mean) = 31.14 Average increase

Comments on the efficacy of the treatment are as follows:

Patient A: I have had noticeable improvement since having the treatment. No pain and at times I forget I have any issues with my knee. The muscle definition has also improved.

Patient B: Great improvement, it's hard to remember how much it hurt before.

Patient C: Every day I feel stronger with gradual improvement to my hips, I believe that I am 100% better now than what I was before starting the treatment and for that I sincerely thank you

Patient D: Seems to be improving steadily. Less than 25% of the pain I experienced prior to the injection. I acknowledge that it will never be as good as when I was young and I will always have some knee pain, but the improvement since the injections is fantastic.

Patient E: Very happy with how knees are, much improvement.

Patient F: A vast improvement in both my knees since my last injection in April. I have a little bit of swelling and pain is minimal but find that climbing stairs and getting off vehicles are hard for me and cannot walk too far.

Patient F: Knee shows definite improvement compared to my untreated knee. Better range of movement and less pain.

Patient G: I was taking anti-inflammatory pills nearly every day before the treatment and now I take one or two a week depending on what activities I am doing. Patient H: Since the injections, I have had a lot more flexibility in the knee. A lot easier to straighten, best I have been able to do for years. The knee hurts occasionally, but only for a second or two, then goes away. The night pain has completely gone. Very happy with the results.

Patient I: Pain becoming less frequent, but am still wearing flat shoes and have begun cycling, swimming and gym work to strengthen muscles.

Claims

CLAIMS:

1 . A method of producing a therapeutically active protein in a blood sample, the method comprising incubating a blood sample in a blood collection or storage device containing one or more substrates, the one or more substrates having a density less than that of erythrocytes and greater than that of plasma and being capable of floating in the blood sample.

2. A method of producing a therapeutically active protein in a blood sample, the method comprising incubating the blood sample in a blood collection or storage device containing one or more substrates, the one or more substrates comprising silicates material.

3. A method of producing a therapeutically active protein in a blood sample, the method comprising incubating the blood sample in a blood collection or storage device containing one or more substrates, the one or more substrates comprising material capable of adhesion to one or more blood cells.

4. The method of claim 1 , claim 2 or claim 3, in which the substrate comprises silicates material with a density between 1025 and 1 125 g/ml at 25°C and atmospheric pressure.

5. The method of claim 1 , claim 2 or claim 3, in which the substrate comprises plastics material, polystyrene, ABS, polyamide or silicates material comprising minerals or other anionic species.

6. The method of claim 6 in which the blood sample is incubated at a temperature of 20 to 40°C for 0.5 to 40 hours or more.

7. An apparatus for producing a therapeutically active protein in a blood sample, the apparatus comprising a blood collection or storage device containing one or more substrates, the one or more substrates (a) having a density less than that of erythrocytes and greater than that of plasma and being capable of floating in the blood sample, (b) comprising silicates material or (c) comprising material capable of adhesion to one or more blood cells.

8. A method for treating a subject in need thereof with a therapeutic protein, the method comprising incubating a blood sample in the blood collection or storage device of claim 7 for a time sufficient to produce a therapeutic protein and administering the blood sample to the subject.

9. The method of claim 8 in which the subject has rheumatism, osteoarthritis and/or a back disorder or complaint.

10. The apparatus of claim 7, in which the therapeutic protein is !1_-1β, IL~1 Ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, !L-15, IL-17, Eotaxin 1 , FGFj3, VEGF, G-CSF, GM-CSF, PDGF, MIP-1<x. ΜΙΡ-1β, CP-1 , !FN-γ, IP-10, RANTES, TNF-a and/or one or more immunoglobulins

11. The method of any one of c!aims 1 , 2, 3 or 8 in which the therapeutic protein is !L-1j IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-7. IL-8, IL-9, IL-10, IL-12, IL-13, SL-15, IL-17, Eotaxin 1 , FGF , VEGF, G-CSF, GM-CSF, PDGF, MIFMa, ΜΙΡ-1β, MCP-1, IFN-γ, IP-10, RANTES, TNF-a and/or one or more immunoglobulins.

12. A method of increasing the cytokine and immunoglobulin content of a blood sample, the method comprising incubating or storing the blood sample in the blood collection or storage vessel of claim 7.

13. A treated blood sample from a subject, the treated blood sample comprising elevated levels of one or more therapeutically active proteins compared to a blood sample from the subject, in which the treated blood sample is provided by incubating or storing the blood sample in the apparatus of claim 7 for such a time to elevate levels of one or more therapeutically active proteins in the sample.

14. A pharmaceutical or veterinary formulation comprising one or more therapeutic protein(s) produced by the method of any one of claims 1, 2, 3 or 8.