Injectable formulation

Abstract

A pharmaceutically acceptable formulation comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein; a buffering agent; a stabilising agent; and a pharmaceutically acceptable diluent or carrier.

Description

[0001] The present invention relates to highly concentrated, stable pharmaceutical formulations of a pharmaceutically active glycosylated protein of the present invention for injection, suitably for subcutaneous injection. Such formulations may comprise, in addition to the high amounts of glycosylated protein, a buffering agent, a stabilizer and a suitable carrier. The invention also relates to a process for the preparation of the said formulation and to the uses of such formulation.

[0002] The pharmaceutical use of antibodies has increased over the past years. In many instances such antibodies are injected via the intravenous (IV) route. Unfortunately, the amount of antibody that can be injected via the intravenous route is limited by the physico-chemical properties of the antibody, in particularly by its solubility and stability in a suitable liquid formulation and by the volume of the infusion fluid. Alternative administration pathways are subcutaneous or intramuscular injection. These injection pathways require high protein concentration in the final solution to be injected [ Shire, S.J., Shahrokh, Z. et al., "Challenges in the development of high protein concentration formulations", J. Pharm. Sci. 2004; 93(6): 1390-1402 ; Roskos, L.K., Davis C.G. et al., "The clinical pharmacology of therapeutic antibodies", Drug Development Research 2004; 61 (3): 108-120 ],

[0003] While antibody formulations have been found suitable for intravenous administration there is a desire to provide highly concentrated, stable pharmaceutical formulations of therapeutically active antibodies for subcutaneous injection. The advantage of subcutaneous injections is that it allows the medical practitioner to perform it in a rather short intervention with the patient. Moreover the patient can be trained to perform the subcutaneous injection by himself. Such selfadministration is particularly useful during maintenance dosing because no hospital care is needed (reduced medical resource utilization). Usually, injections via the subcutaneous route are limited to approximately 2 ml. For patients requiring multiple doses, several unit dose formulations can be injected at multiple sites of the body surface.

[0004] The injection of parenteral drugs into the hypodermis is generally limited to volumes of less than 2 ml due to the viscoelastic resistance to hydraulic conductance in the subcutaneous (SC) tissue, due to the generated backpressure upon injection [ Aukland K. and Reed R., "Interstitial- Lymphatic Mechanisms in the control of Extracellular Fluid Volume", Physiology Reviews", 1993; 73:1-78 ], as well as due to the perceptions of pain.

[0005] The preparation of high concentration protein formulations is rather challenging and there is a need to adapt each formulation to the particular proteins used because each protein has a different aggregation behaviour. Aggregates are suspected to cause immunogenicity of therapeutic proteins in at least some of the cases. Immunogenic reaction against protein or antibody aggregates may lead to neutralizing antibodies which may render the therapeutic protein or antibody ineffective. It appears that the immunogenicity of protein aggregates is most problematic in connection with subcutaneous injections, whereby repeated administration increases the risk of an immune response.

[0006] While antibodies have a very similar overall structure, such antibodies differ in the amino acid composition (in particular in the CDR regions responsible for the binding to the antigen) and the glycosylation pattern. Moreover, there may additionally be post-translational modifications such as charge and glycosylation variants.

[0007] The protein of the present invention is a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein.

[0008] The glycosylated protein described in the present disclosure is in development for a range of indications. Glycosylated proteins are typically produced by fermentation and expression from a cell line. When glycosylated proteins are expressed from a cell it goes through a process called post translational modification. Sugars attach themselves to side chains on the glycosylated protein. There can be variation in the glycosylated protein depending on the process by which the peptide has been expressed. Factors can include the cell from which the glycosylated protein is expressed and the environmental conditions such as nutrients, oxygen levels and lysis.

[0009] The present disclosure relates to a glycosylated form of the protein described in WO2015 / 040398. The application describes the protein and a method of manufacture.

[0010] The protein is disclosed as Sequence ID No.2

[0011] WO2013 / 136078 describes the use of p75NTR neurotrophin binding protein in the treatment of pain.

[0012] W02016 / 009222 discusses the use of p75NTR neurotrophin binding protein in the treatment of osteoarthritis. Treatment includes curative or reversal of the disease as well as relief from symptoms and reflects disease modifying effect.

[0013] Additional applications disclose preferred forms and methods for manufacture of the glycosylated protein of the invention.

[0014] The current invention relates to a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein. The p75NTR(NBP)-Fc fusion protein finds use in the treatment of pain and other neurotrophic factor related pathologies such as psoriasis, eczema, rheumatoid arthritis, cystitis, endometriosis and osteoarthritis. In its glycosylated form it can be administered once a month and demonstrate long lasting efficacy, however, it’s difficult to formulate such a molecule appropriately. The problem to be solved by the present invention is to provide novel highly concentrated, stable pharmaceutical formulations of the glycosylated protein of the present invention for injection, preferably subcutaneous injection. Such formulations comprise, in addition to the high amounts of the glycosylated protein, a buffering agent, a stabilizer and a pharmaceutically acceptable diluent or carrier.

[0015] The preparation of highly-concentrated antibody formulations is challenging because of a potential increase in viscosity at higher protein concentration and a potential increase in protein aggregation, a phenomenon that is per se concentration-dependent. High viscosities negatively impact the process ability (e.g. pumping and filtration steps) of the antibody formulations and the administration (e.g. the syringe ability). By the addition of excipients high viscosities could be decreased in some cases. Control and analysis of protein aggregation is an increasing challenge. Aggregation is potentially encountered during various steps of the manufacturing process, which include fermentation, purification, formulation and during storage. Different factors, such as temperature, protein concentration, agitation stress, freezing and thawing, solvent and surfactant effects, and chemical modifications, might influence the aggregation behaviour of a therapeutic protein. During development of a highly concentrated antibody formulation the aggregation tendency of the protein has to be monitored and controlled by the addition of various excipients and surfactants [ Kiese S. et al., J. Pharm. Sci., 2008; 97(10); 4347-4366 ].

[0016] Figures

[0017] Fig 1 references Seq ID No. 1 , the amino acid sequence for the glycosylated protein of the present disclosure.

[0018] Fig 2 references Seq ID No. 2, a sequence for the P75NTR(NBP) portion of the glycosylated protein of the present invention.

[0019] In a first aspect the present invention provides

[0020] A stable pharmaceutically acceptable formulation, suitable for injection comprising: a) 10-360mg / ml of a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising a p75NTR(NBP) portion, having at least 85% sequence identity with Seq ID No. 2; and an immunoglobulin Fc portion, wherein, the p75NTR(NBP) and Fc portions are connected via a linker, the linker comprises a peptide of formula Gx, where x is 1 , 2, 3, 4, 5 or 6_wherein the linker does not comprise or consist of the sequence GGGGS; b) A buffering agent c) A stabilising agent and a pharmaceutically acceptable diluent or carrier.

[0021] Suitably the glycosylated protein linker portion is GGG and the Fc is a human Fc Suitably the P75NTR(NBP) portion of glycosylated p75NTR neurotrophin binding protein (NBP)-

[0022] Fc fusion protein has at least 85% sequence identity with the protein of Seq ID No. 2

[0023] Suitably the P75NTR(NBP) portion of glycosylated p75NTR neurotrophin binding protein (NBP)-

[0024] Fc fusion protein has at least 90% sequence identity with the protein of Seq ID No. 2

[0025] Suitably the P75NTR(NBP) portion of glycosylated p75NTR neurotrophin binding protein (NBP)-

[0026] Fc fusion protein has at least 95% sequence identity with the protein of Seq ID No. 2

[0027] Suitably the P75NTR(NBP) portion of glycosylated p75NTR neurotrophin binding protein (NBP)-

[0028] Fc fusion protein has at least 97% sequence identity with the protein of Seq ID No. 2

[0029] Suitably the P75NTR(NBP) portion of glycosylated p75NTR neurotrophin binding protein (NBP)- Fc fusion protein comprises Seq ID No. 2.

[0030] Suitably the glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein has at least 85% sequence identity with the protein of Seq ID No. 1

[0031] Suitably the glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein has at least 90% sequence identity with the protein of Seq ID No. 1

[0032] Suitably the glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein has at least 95% sequence identity with the protein of Seq ID No. 1

[0033] Suitably the glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein has at least 97% sequence identity with the protein of Seq ID No. 1

[0034] Suitably the glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein comprises Seq ID No. 1 .

[0035] Suitably, the carrier is saline.

[0036] In one embodiment of the invention the formulation is suitable for injection.

[0037] In one embodiment of the invention the formulation is suitable for subcutaneous injection.

[0038] In one embodiment of the invention the volume of the formulation does not exceed 2ml.

[0039] Suitably the volume of the subcutaneously injected formulation does not exceed 2ml.

[0040] In a further aspect the present invention provides for use of a formulation for the preparation of a medicament useful for treating Pain or osteoarthritis in a subject comprising administering the formulation described herein to a subject in an amount effective to treat the said disease or disorder.

[0041] In another aspect the present invention there are provided methods of treating Pain or Osteoarthritis in a subject comprising administering the formulation described herein to a subject in an amount effective to treat the said disease or disorder.

[0042] The present invention also provides pharmaceutical compositions of the present invention in the form of a kit comprising both injection components and suitable instructions for their subcutaneous administration.

[0043] The formulation of the present invention may be provided in liquid form or may be provided in lyophilized form. The antibody concentration in the reconstituted formulation can be increased by reconstitution of a lyophilized formulation to provide a protein concentration in the reconstituted formulation which is about 2-40 times greater than the protein concentration in the mixture before the lyophilization step.

[0044] The concentration of the glycosylated protein described herein, in the formulation is 10 to 180 mg / ml, suitably 160mg / ml.

[0045] In one embodiment the concentration of the glycosylated protein described herein, in the formulation is 10-20 mg / ml

[0046] In one embodiment the concentration of the glycosylated protein described herein, in the formulation is 20-130 mg / ml

[0047] In one embodiment the concentration of the glycosylated protein described herein, in the formulation is 30-120 mg / ml

[0048] In one embodiment the concentration of the glycosylated protein described herein, in the formulation is 40-110 mg / ml

[0049] In one embodiment the concentration of the glycosylated protein described herein, in the formulation is 40-110 mg / ml

[0050] In one embodiment the concentration of the glycosylated protein described herein, in the formulation is 40-100 mg / ml

[0051] In one embodiment the concentration of the glycosylated protein described herein, in the formulation is 50-100 mg / ml

[0052] The pH of the formulation is 6.5+ / -2.0. Suitably the pH is 5.5-7. Suitably the pH is 5.5-6.5. Suitably the pH is 5.9, 6.0, 6.1 , 6.2. 6.3, 6.4 or 6.5.

[0053] The concentration of the buffering agent providing a pH of 6.5 ± 2.0 is 1 to 50 mM, suitably 10 to 35 mM or suitably 25 mM. Various buffering agents are known to the person skilled in the art as outlined further below. Suitable buffering agents can include histidine, acetate, phosphate or glutamate buffers.

[0054] Suitably the buffering agent can be a histidine buffer, e.g. L-histidine / HCl. In a particular embodiment the pH of the L-histidine / HCI buffer is about 5.5 or about 6.0. Suitably the concentration of the L-histidine / L-histidine / HCI buffer is 25mM.

[0055] The stabilizer (used synonymously with the term "stabilizing agent" in the present patent description) is e.g. a carbohydrate or saccharide or a sugar admitted by the authorities as a suitable additive or excipient in pharmaceutical formulations, e.g. a,a-trehalose dihydrate or sucrose or mannitol. Suitably the stabiliser is mannitol. The concentration of the stabilizer is 15 to 250 mM, or 150 to 250 mM, or about 200 mM.

[0056] The formulation may contain suitable additional agents. Such agents include without limitation surfactants, amino acids, salts and hyaluronidase enzymes.

[0057] Suitable amino acids for inclusion include L-Arg-HCI, Glycine & Proline.

[0058] Suitable amino acids are L-Arg-HCI

[0059] A suitable diluent or carrier for the formulation could include NaCI solution, L-Arg-HCI.

[0060] Suitably the diluent or carrier is NaCI solution. Suitably the concentration is 25-125mM. Suitably the concentration is 40-1 OOmM. Suitably the concentration is 50mM

[0061] The formulation may optionally contain a suitable pharmaceutical surfactant or surfactants. Suitable examples of pharmaceutically acceptable surfactants include polyoxyethylen-sorbitan fatty acid esters (Tween), polyethylene-polypropylene glycols, polyoxyethylene-stearates, polyoxyethylene alkyl ethers, e.g. polyoxyethylene monolauryl ether, alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulphate (SDS). Most suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Most suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Most suitable polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Most suitable alkylphenolpolyoxyethylene ethers are sold under the trade name Triton-X. The nonionic surfactant can be a polysorbate, e.g. selected from the group of polysorbate 20, polysorbate 80 and polyethylene-polypropylene copolymer. The concentration of the nonionic surfactant is 0.01 to 0.1 % (w / v), or 0.01 to 0.08 % (w / v), or 0.025 to 0.075 % (w / v), or more particularly about 0.02, 0.04 or 0.06 % (w / v).

[0062] Suitably, the formulation may optionally contain a hyaluronidase enzyme. The concentration of the hyaluronidase enzyme depends on the actual hyaluronidase enzyme used in the preparation of the formulation in accordance with the invention. An effective amount of the hyaluronidase enzyme can easily be determined by the person skilled in the art based on the disclosure further below. It should be provided in sufficient amount so that an increase in the dispersion and absorption of the glycosylated protein of the invention is possible. The minimal amount of the hyaluronidase enzyme is > 150 U / ml. More particularly the effective amount of the hyaluronidase enzyme is about 1 '000 to 16'000 U / ml, whereby the said amount corresponds to about 0.01 mg to 0.16 mg protein based on an assumed specific activity of 100'000 U / mg. Alternatively the concentration of the hyaluronidase enzyme is about 1'500 to 12'000 U / ml, or more particularly about 2'000 U / ml or about 12'000 U / ml. The amounts specified hereinbefore correspond to the amount of hyaluronidase enzyme initially added to the formulation. As evidenced in the example formulations the hyaluronidase enzyme concentrations measured in the final formulation may vary within a certain range. Thus, e.g. the actually measured hyaluronidase enzyme (HE) concentration measured just after adding 12'000 U / ml of enzyme showed variations between 12355 U / ml to 15178 U / ml (see Tables 1 Formulations A to F and Table 3 Formulation H). The hyaluronidase enzyme is present either as a combined final formulation or for use for coadministration, e.g. as a co-formulation.

[0063] The hyaluronidase enzyme may be derived from animals, human samples or manufactured based on the recombinant DNA technology as described further below.

[0064] It has been proposed to facilitate the subcutaneous injection of therapeutic proteins and antibodies by using small amounts of soluble hyaluronidase glycoproteins (sHASEGPs); see W02006 / 091871 . It has been shown that the addition of such soluble hyaluronidase glycoproteins (either as a combined formulation or by co-administration) facilitates the administration of therapeutic drug into the hypodermis. By rapidly depolymerizing hyaluronan HA in the extracellular space sHASEGP reduces the viscosity of the interstitium, thereby increasing hydraulic conductance and allowing for larger volumes to be administered safely and comfortably into the subcutaneous tissue. The increased hydraulic conductance induced by sHASEGP through reduced interstitial viscosity allows for greater dispersion, potentially increasing the systemic bioavailability of SC administered therapeutic drug.

[0065] The highly concentrated, stable pharmaceutical formulations of the present invention comprising a soluble hyaluronidase glycoprotein are therefore particularly suited for subcutaneous injection. It is clearly understood by the person skilled in the art that such a formulation comprising a glycosylated protein of the present invention and a soluble hyaluronidase glycoprotein can be provided for administration in form of one single combined formulation or alternatively in form of two separate formulations which can be mixed just prior to the subcutaneous injection. Alternatively the glycosylated protein of the present invention and the soluble hyaluronidase glycoprotein can be administered as separate injections at different sites of the body, preferably at sites which are immediately adjacent to each other. It is also possible to inject the therapeutic agents present in the formulation in accordance with the present invention as consecutive injections, e.g. first the soluble hyaluronidase glycoprotein followed by the injection of the antiantibody formulation. These injections can also be performed in the reversed order, viz. by first injecting the glycosylated protein of the present invention formulation followed by injecting the soluble hyaluronidase glycoprotein.

[0066] A further aspect of the present invention relates to injection devices comprising a formulation in accordance with the present invention.

[0067] A number of a soluble hyaluronidase glycoprotein are known in the prior art. In order to further define the function, the mechanism of action and the properties of such soluble hyaluronidase glycoproteins the following background information is provided. The SC (hypodermal) interstitial matrix is comprised of a network of fibrous proteins embedded within a viscoelastic gel of glycosaminoglycans. Hyaluronan (HA), a non-sulfated repeating linear disaccharide, is the prominent glycosaminoglycan of the SC tissue. HA is secreted into the interstitium by fibroblasts as a high molecular weight, megadalton viscous polymer that is subsequently degraded locally, in the lymph, and in the liver, through the action of lysosomal hyaluronidases and exoglycosidases. Approximately 50% of the hyaluronan in the body is produced by the SC tissue, where it is found at approximately 0.8 mg / gm wet weight tissue [Aukland K. and Reed R., supra]. It is estimated that the average 70 kg adult contains 15 grams of HA, of which 30 percent is turned over (synthesized and degraded) daily [ Laurent L.B., et al., "Catabolism of hyaluronan in rabbit skin takes place locally, in lymph nodes and liver", Exp. Physiol. 1991 ; 76: 695-703 ]. As a major constituent of the gel-like component of the hypodermal matrix, HA contributes significantly to its viscosity.

[0068] Glycosaminoglycans (GAGs) are complex linear polysaccharides of the extracellular matrix (ECM). GAGs are characterized by repeating disaccharide structures of an N-substituted hexosamine and an uronic acid (in the case of hyaluronan (HA), chondroitin sulfate (CS), chondroitin (C), dermatan sulfate (DS), heparan sulfate (HS), and heparin (H)), or a galactose (in the case of keratan sulfate (KS)). Except for HA, all exist covalently bound to core proteins. The GAGs with their core proteins are structurally referred to as proteoglycans (PGs).

[0069] Hyaluronan (HA) is found in mammals predominantly in connective tissues, skin, cartilage, and in synovial fluid. Hyaluronan is also the main constituent of the vitreous of the eye. In connective tissue, the water of hydration associated with hyaluronan creates hydrated matrices between tissues. Hyaluronan plays a key role in biological phenomena associated with cell motility including rapid development, regeneration, repair, embryogenesis, embryological development, wound healing, angiogenesis, and tumorigenesis ( Toole, Cell Biol. Extracell. Matrix, Hay (ed), Plenum Press, New York, 1991 ; pp. 1384-1386 ; Bertrand et al., Int. J. Cancer 1992; 52:1-6 ; Knudson et ak, FASEB J. 1993; 7:1233-1241 ]. In addition, hyaluronan levels correlate with tumour aggressiveness [ Ozello et al., Cancer Res. 1960; 20:600-604 ; Takeuchi et al., Cancer Res. 1976; 36:2133-2139 ; Kimata et al., Cancer Res. 1983; 43:1347-1354 ],

[0070] HA is found in the extracellular matrix of many cells, especially in soft connective tissues. HA has been assigned various physiological functions, such as in water and plasma protein homeostasis [ Laurent T.C. et al., FASEB J., 1992; 6: 2397-2404 ]. HA production increases in proliferating cells and may play a role in mitosis. It has also been implicated in locomotion and cell migration. HA seems to play important roles in cell regulation, development, and differentiation [Laurent et al., supra].

[0071] HA has widely been used in clinical medicine. Its tissue protective and rheological properties have proved useful in ophthalmic surgery (e.g. to protect the corneal endothelium during cataract surgery). Serum HA is diagnostic of liver disease and various inflammatory conditions, such as rheumatoid arthritis. Interstitial edema caused by accumulation of HA may cause dysfunction in various organs [Laurent et al., supra].

[0072] Hyaluronan protein interactions also are involved in the structure of the extracellular matrix or "ground substance".

[0073] Hyaluronidases are a group of generally neutral- or acid-active enzymes found throughout the animal kingdom. Hyaluronidases vary with respect to substrate specificity, and mechanism of action ( WO 2004 / 078140 ). There are three general classes of hyaluronidases:

[0074] • 1 . Mammalian-type hyaluronidases, (EC 3.2.1 .35) which are endo-beta-N- acetylhexosaminidases with tetrasaccharides and hexasaccharides as the major end products. They have both hydrolytic and transglycosidase activities, and can degrade hyaluronan and chondroitin sulfates (CS), generally C4-S and C6-S.

[0075] • 2. Bacterial hyaluronidases (EC 4.2.99.1) degrade hyaluronan and, and to various extents, CS and DS. They are endo-beta-N-acetylhexosaminidases that operate by a beta elimination reaction that yields primarily disaccharide end products.

[0076] • 3. Hyaluronidases (EC 3.2.1 .36) from leeches, other parasites, and crustaceans are endo-beta-glucuronidases that generate tetrasaccharide and hexasaccharide end products through hydrolysis of the beta 1-3 linkage.

[0077] Mammalian hyaluronidases can be further divided into two groups: neutral-active and acid-active enzymes. There are six hyaluronidase-like genes in the human genome, HYAL1 , HYAL2, HYAL3, HYAL4, HYALP1 and PH20 / SPAM1. HYALP1 is a pseudogene, and HYAL3 has not been shown to possess enzyme activity toward any known substrates. HYAL4 is a chondroitinase and exhibits little activity towards hyaluronan. HYAL1 is the prototypical acidactive enzyme and PH20 is the prototypical neutral-active enzyme. Acid-active hyaluronidases, such as HYAL1 and HYAL2 generally lack catalytic activity at neutral pH (i.e. pH 7). For example, HYAL1 has little catalytic activity in vitro over pH 4.5 [ Frost LG. and Stem, R., "A microtiterbased assay for hyaluronidase activity not requiring specialized reagents", Anal. Biochemistry, 1997; 251 :263-269 ]. HYAL2 is an acid-active enzyme with a very low specific activity in vitro.

[0078] The hyaluronidase-like enzymes can also be characterized by those which are generally locked to the plasma membrane via a glycosylphosphatidyl inositol anchor such as human HYAL2 and human PH20 [ Danilkovitch-Miagkova et al., Proc. Natl. Acad. Sci. U SA, 2003; 100(8):4580- 4585 ; Phelps et ak, Science 1988; 240(4860): 1780-1782 ], and those which are generally soluble such as human HYAL1 [ Frost, LG. et ak, "Purification, cloning, and expression of human plasma hyaluronidase", Biochem. Biophys. Res. Commun. 1997; 236(1): 10-15 ]. However, there are variations from species to species: bovine PH20 for example is very loosely attached to the plasma membrane and is not anchored via a phospholipase sensitive anchor [ Lalancette et al., Biol. Reprod., 2001 ; 65(2):628- 36 ]. This unique feature of bovine hyaluronidase has permitted the use of the soluble bovine testes hyaluronidase enzyme as an extract for clinical use (Wydase™, Hyalase™). Other PH20 species are lipid anchored enzymes that are generally not soluble without the use of detergents or lipases. For example, human PH20 is anchored to the plasma membrane via a GPI anchor. Attempts to make human PH20 DNA constructs that would not introduce a lipid anchor into the polypeptide resulted in either a catalytically inactive enzyme, or an insoluble enzyme [ Arming et al., Eur. J. Biochem., 1997; 247(3):810-4 ]. Naturally occurring macaque sperm hyaluronidase is found in both a soluble and membrane bound form. While the 64 kDa membrane bound form possesses enzyme activity at pH 7.0, the 54 kDa form is only active at pH 4.0 [ Cherr et al., Dev. Biol., 1996; 10; 175(1): 142-53 ]. Thus, soluble forms of PH20 are often lacking enzyme activity under neutral conditions.

[0079] As noted above and in accordance with the teachings in W02006 / 091871 small amounts of soluble hyaluronidase glycoproteins (sHASEGPs) can be introduced into a formulation in order to facilitate the administration of therapeutic drug into the hypodermis. By rapidly depolymerizing HA in the extracellular space sHASEGP reduces the viscosity of the interstitium, thereby increasing hydraulic conductance and allowing for larger volumes to be administered safely and comfortably into the SC tissue. The increased hydraulic conductance induced by sHASEGP through reduced interstitial viscosity allows for greater dispersion, potentially increasing the systemic bioavailability of SC administered therapeutic drug.

[0080] When injected in the hypodermis, the depolymerization of HA by sHASEGP is localized to the injection site in the SC tissue. Experimental evidence shows that the sHASEGP is inactivated locally in the interstitial space with a half life of 13 to 20 minutes in mice, without detectable systemic absorption in blood following single intravenous dose in CD-1 mice. Within the vascular compartment sHASEGP demonstrates a half life of 2.3 and 5 minutes in mice and Cynomolgus monkeys, respectively, with doses up to 0.5 mg / kg. The rapid clearance of sHASEGP, combined with the continual synthesis of the HA substrate in the SC tissue, results in a transient and locally-active permeation enhancement for other co-injected molecules, the effects of which are fully reversible within 24 to 48 hours post administration [ Bywaters G.L., et al., "Reconstitution of the dermal barrier to dye spread after Hyaluronidase injection", Br. Med. J., 1951 ; 2 (4741): 1178-1183 ],

[0081] In addition to its effects on local fluid dispersion, sHASEGP also acts as absorption enhancer. Macromolecules greater than 16 kilodaltons (kDa) are largely excluded from absorption through the capillaries via diffusion and are mostly absorbed via the draining lymph nodes. A subcutaneously administered macromolecule such as e.g. a therapeutic antibody (molecular weight approximately 150 kDa) must therefore traverse the interstitial matrix before reaching the draining lymphatics for subsequent absorption into the vascular compartment. By increasing local dispersion, sHASEGP increases the rate (Ka) of absorption of many macromolecules. This leads to increased peak blood levels (Cmax ) and potentially to increased bioavailability relative to SC administration in the absence of sHASEGP [ Bookbinder L.H., et al., "A recombinant human enzyme for enhanced interstitial transport of therapeutics", J. Control. Release 2006; 114: 230- 241 ]. Hyaluronidase products of animal origin have been used clinically for over 60 years, primarily to increase the dispersion and absorption of other co-administered drugs and for hypodermoclysis (SC injection / infusion of fluid in large volume) [ Frost G.I., "Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration", Expert Opinion on Drug Delivery, 2007; 4: 427-440 ]. The details on the mechanism of action of hyaluronidases have been described in detail in the following publications: Duran-Reynolds F., "A spreading factor in certain snake venoms and its relation to their mode of action", CR Soc Biol Paris, 1938; 69-81 ; Chain E., "A mucolytic enzyme in testes extracts", Nature 1939; 977-978 ; Weissmann B., "The transglycosylative action of testicular hyaluronidase", J. Biol. Chem., 1955; 216: 783-94 ; Tammi, R., Saamanen, A.M., Maibach, H.I., Tammi M., "Degradation of newly synthesized high molecular mass hyaluronan in the epidermal and dermal compartments of human skin in organ culture", J. Invest. Dermatol. 1991 ; 97:126-130 ; Laurent, U.B.G., Dahl, L.B., Reed, R.K., "Catabolism of hyaluronan in rabbit skin takes place locally, in lymph nodes and liver", Exp. Physiol. 1991 ; 76: 695-703 ; Laurent, T.C. and Fraser, J.R.E., "Degradation of Bioactive Substances: Physiology and Pathophysiology", Henriksen, J.H. (Ed) CRC Press, Boca Raton, FL; 1991. pp. 249-265 ; Harris, E.N., et ak, "Endocytic function, glycosaminoglycan specificity, and antibody sensitivity of the recombinant human 190-kDa hyaluronan receptor for endocytosis (HARE)", J. Biol. Chem. 2004; 279:36201-36209 ; Frost, G.I., "Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration", Expert Opinion on Drug Delivery, 2007; 4: 427-440 . Hyaluronidase products approved in EU countries include Hylase® "Dessau" and Hyalase® Hyaluronidase products of animal origin approved in the US include Vitrase™, Hydase™, and Amphadase™.

[0082] The safety and efficacy of hyaluronidase products have been widely established. The most significant safety risk identified is hypersensitivity and / or allergenicity, which is thought to be related to the lack of purity of the animal-derived preparations [ Frost, G.I., "Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration", Expert Opinion on Drug Delivery, 2007; 4 : 427-440 ]. It should be noted that there are differences with respect to the approved dosages of animal-derived hyaluronidases between the UK, Germany and the US. In the UK, the usual dose as an adjuvant to subcutaneous or intramuscular injection is 1500 units, added directly to the injection. In the US, the usual dose used for this purpose is 150 units. In hypodermoclysis, hyaluronidase is used to aid the subcutaneous administration of relatively large volumes of fluids. In the UK, 1500 units of hyaluronidase are generally given with each 500 to 1000 ml of fluid for subcutaneous use. In the US, 150 units are considered adequate for each liter of hypodermoclysis solution. In Germany, 150 to 300 units are considered adequate for this purpose. In the UK, the diffusion of local anesthetics is accelerated by the addition of 1500 units. In Germany and the US 150 units are considered adequate for this purpose. The dosage differences notwithstanding (the dosage in the UK is ten times higher than in the US), no apparent differences in the safety profiles of animal-derived hyaluronidase products marketed in the US and UK, respectively, have been reported. On December 2, 2005, Halozyme Therapeutics Inc. received approval from the FDA for an injectable formulation of the recombinant human hyaluronidase, rHuPH20 (HYLENEX™). The FDA approved HYLENEX™ at a dose of 150 units for SC administration of the following indications:

[0083] • as an adjuvant to increase the absorption and dispersion of other injected drugs

[0084] • for hypodermoclysis

[0085] • as an adjunct in SC urography for improving resorption of radiopaque agents.

[0086] As part of that regulatory review it was established that rHuPH20 possesses the same properties of enhancing the dispersion and absorption of other injected drugs as the previously approved animal-derived hyaluronidase preparations, but with an improved safety profile. In particular, the use of recombinant human hyaluronidase (rHuPH20) compared with animal-derived hyaluronidases minimizes the potential risk of contamination with animal pathogens and transmissible spongiform encephalopathies.

[0087] Detailed experimental work as outlined has shown that the formulations of the present invention surprisingly have favourable storage stability, low volume, appropriate delivery characteristics and drug release profile and fulfil all necessary requirements for approval by the health authorities.

[0088] A number of suitable hyaluronidase enzymes in accordance with the present invention are known from the prior art. The preferred enzyme is a human hyaluronidase enzyme, most preferably the enzyme known as rHuPH20. rHuPH20 is a member of the family of neutral and acid-active p-1 ,4 glycosyl hydrolases that depolymerize hyaluronan by the hydrolysis of the p-1 ,4 linkage between the C1 position of N-acetyl glucosamine and the C4 position of glucuronic acid. Hyaluronan is a polysaccharide found in the intracellular ground substance of connective tissue, such as the subcutaneous interstitial tissue, and of certain specialized tissues, such as the umbilical cord and vitreous humor. The hydrolysis of hyaluronan temporarily decreases the viscosity of the interstitial tissue and promotes the dispersion of injected fluids or of localized transudates or exudates, thus facilitating their absorption. The effects of hyaluronidase are local and reversible with complete reconstitution of the tissue hyaluronan occurring within 24 to 48 hours [ Frost, G.I., "Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration", Expert Opinion on Drug Delivery, 2007; 4:427-440 ]. The increase in the permeability of connective tissue through the hydrolysis of hyaluronan correlates with the efficacy of hyaluronidase for their capability to increase the dispersion and absorption of co-administered molecules.

[0089] The human genome contains several hyaluronidase genes. Only the PH20 gene product possesses effective hyaluronidase activity under physiologic extracellular conditions and acts as a spreading agent, whereas acid-active hyaluronidases do not have this property. rHuPH20 is the first and only recombinant human hyaluronidase enzyme currently available for therapeutic use. Naturally occurring human PH20 protein has a lipid anchor attached to the carboxy terminal amino acid that anchors it to the plasma membrane. The rHuPH20 enzyme developed by Halozyme is a truncated deletion variant that lacks such amino acids in the carboxy terminus responsible for the lipid attachment. This gives rise to a soluble, neutral pH- active enzyme similar to the protein found in bovine testes preparations. The rHuPH20 protein is synthesized with a 35 amino acid signal peptide that is removed from the N-terminus during the process of secretion. The mature rHuPH20 protein contains an authentic N-terminal amino acid sequence orthologous to that found in some bovine hyaluronidase preparations.

[0090] The PH20 hyaluronidases, including the animal derived PH20 and recombinant human rHuPH20, depolymerize hyaluronan by the hydrolysis of the p-1 ,4 linkage between the C1 position of N- acetyl glucosamine and the C4 position of glucuronic acid. The tetrasaccharide is the smallest digestion product [ Weissmann, B., "The transglycosylative action of testicular hyaluronidase", J. Biol. Chem., 1955; 216: 783-94 ]. This N-acetyl glucosamine / glucuronic acid structure is not found in N-linked glycans of recombinant biological products and therefore rHuPH20 will not affect the glycosylation of antibodies it is formulated with. The rHuPH20 enzyme itself possesses six N-linked glycans per molecule with core structures similar to that found in monoclonal antibodies. As anticipated, these N-linked structures do not change over time, confirming the lack of enzymatic activity of rHuPH20 on these N-linked glycan structures. The short half life of rHuPH20 and the constant synthesis of hyaluronan lead to a short and local action of the enzyme on tissues.

[0091] The hyaluronidase enzyme which is an excipient in the subcutaneous formulation in accordance with the present invention can be prepared by using recombinant DNA technology. In this way it is ensured that the same protein (identical amino acid sequence) is obtained all the time and that an allergic reaction, e.g. caused by contaminating proteins co-purified during extraction from a tissue, is avoided. The hyaluronidase enzyme used in the formulation as exemplified herein is a human enzyme, viz. rHuPH20.

[0092] The amino acid sequence of rHuPH20 (HYLENEX™) is well known and available under CAS Registry No. 75971-58-7. The approximate molecular weight is 61 kDa.

[0093] Multiple structural and functional comparisons have been performed between naturally sourced mammalian hyaluronidase and PH-20 cDNA clones from humans and other mammals. The PH- 20 gene is the gene used for the recombinant product rHuPH20; however the recombinant drug product is a 447 amino acid truncated version of the full protein encoded by the PH-20 gene. Structural similarities with respect to amino acid sequences rarely exceed 60% in any comparison. Functional comparisons show that the activity of rHuPH20 is very similar to that of previously approved hyaluronidase products. This information is consistent with the clinical findings during the past 50 years that regardless of the source of the hyaluronidase, the clinical safety and efficacy of units of hyaluronidase are equivalent. The glycosylated protein of the present invention may be made by the following process:

[0094] Turning to the process by which the glycosylated protein is expressed, methods for expressing glycosylated proteins are well known to those skilled in the art. Such processes typically go through a number of stages; inoculation, fermentation and harvesting. The, cells, feeds and media used may be obtained from the company ‘Lonza’. Addressing each individually:

[0095] Vector Construction

[0096] The process starts with codon optimisation of the DNA sequence to allow efficient expression in CHO cell lines with a signal sequence at the N-terminus that directs the protein for secreted expression and appropriate restriction enzyme sites to enable cloning into cloning into the glutamine synthetase (GS) vectors and a Kozak sequence between the 5’ restriction enzyme site and the ‘ATG’ start codon. These sequences were submitted to and synthesised by Life Technologies and provided within a cloning vector. Other vectors with different secretion sequences and a different selectable marker (example dihydrofolate reductase (DHFR) instead of GS). The 1405 base pair (bp) DNA fragment encoding SEQ ID No 1 was removed from the cloning vector into the GS expression vector pXC-17 via Hindlll and EcoR1 digestion. The digested products (SEQ ID No 1 DNA fragment and vector were ligated and the ligated products used to transform chemically competent Escherichia Coli (E. coli) cells (for example TOP10 cells). Single colonies were analysed for the correct insert and one positive clone was selected and plasmid DNA prepared and sequence (in forward reverse orientation) to ensure the correct sequence was present. The plasmid was renamed p SEQ ID No 1 / SGV. Sufficient linearised DNA for the cell line construction process was generated by linearisation of p SEQ ID No 1 / SGV with the restriction enzyme Pvul.

[0097] Lonza Biologies’ mammalian Chinese Hamster Ovary (CHO) K1 SV Glutamine Synthetase - Knock Out (GS-KO) expression system was used to produce SEQ ID No 1. The CHOK1 SV GS- KO host cell line is a derivative of the CHOK1 SV host cell line with the endogenous gene for GS ‘knocked out’. The host cell line was derived from Lonza Biologies’ CHOK1SV GS-KO host working cell bank designated the code 760-W (prepared from the master host cell bank 760-M).

[0098] Other suitable CHO Cell lines include for example CHO-DGB with a DHFR selectable marker on the expression vector. There are also other GS-CHO knock out cell lines available that could be used (for example CHOSOURCE GS KO (Horizon discovery) and CHOZN (MilliporeSigma). There are also other mammalian systems available (for example PER.C6® human cells (Crucell).

[0099] Transfection by electroporation was performed via a single pulse using linearised plasmid DNA to generate stable CHOK1 SV GS-KO transfectant minipools expressing SEQ ID No 1. CaPO4 or lipid-based reagents (Lipofectamine, Fugene, Transfectin) can also be used to transfect cells. A number of transfections was performed and split into a larger number of minipools. The day after transfection, selective medium containing methionine sulphoximine (MSX) was added to each transfectant minipool. The addition of MSX, which inhibits GS, to the growth medium increases the selective pressure in the transfectant minipools; following cloning this selective pressure is no longer required. If one used the same vector with a CHOK1 SV cell line, the selective pressure would need to be applied throughout the cell line development and included in the early growth steps (before the inoculation of the fermenter).

[0100] After ~14 days of incubation, >500 transfectant minipools are expanded to suspension culture in wells of shaken 96 deep well plates. 3 days after transfer to suspension culture, samples from >500 wells were assayed for protein production using the Octet® method and the product concentration data generated were used to identify the overall highest producer transfectant minipools. Multiple high producing transfectant minipools were combined to generate >4 enriched transfectant pools.

[0101] The enriched transfectant pools were single cell sorted using a fluorescence activated cell sorter (FACS). Following incubation and imaging, supernatant samples from wells identified as containing a single colony were screened for SEQ ID No 1 production. >500 clonal cell lines were subsequently selected for further evaluation in an abridged fed-batch suspension culture productivity screen and transferred to suspension culture in shaken 96 deep well plates. Other methods can be used to derive monoclonal cell lines (for example a two step dilution cloning in 96-well plates or using semi-solid media plates.

[0102] >500 clonal cell lines were successfully adapted to suspension culture and were screened for productivity in a fed batch media. Following this screen, 20 clonal cell lines were selected for further evaluation, based upon their productivity ranking and the parental transfectant pool from which they were derived; these were expanded to culture in shake-flasks and their growth during routine subculture assessed. 9 lead candidate cell lines were selected for progression and cryopreservation of associated research cell banks (RGB). Selection was based on the expression levels of SEQ ID No 1 , acceptable growth characteristics and image evidence that each cell line arose from a single colony.

[0103] A 70-generation cell line stability study was initiated with all 9 lead candidate cell lines in which 2 independently subcultured lineages of each cell line were then established. The growth and productivity characteristics for each lineage were then evaluated at two points (~5 and ~35 generations beyond that of the associated RGBs) in fed-batch miniature bioreactor culture screens designed to mimic the cGMP manufacturing bioreactor culture process. Product from cultures of each of the 9 cell lines evaluated at ~5 generations was also assessed for product quality using a range of assays.

[0104] After a review of all available data, six lead candidate cell lines were selected for continuation in the 70-generation cell line stability study. Once the 6 cell lines had all accrued greater than seventy generations beyond the associated RCB, a final fed-batch miniature bioreactor culture screen was undertaken in which early and late generation cultures were evaluated concurrently.

[0105] Following evaluation of the consistency of the growth, productivity and product characteristics data across the 70-generation study, the lead cell line was chosen. A 200 vial GMP master cell bank was manufactured from a single vial of the relevant RCB and tested in accordance with current regulatory requirements. When sufficient cells were obtained, cells were aliquoted in cryopreservation medium (92.5 % CM66 ! 7.5% DMSO) into polypropylene vials (each containing approximately 1 .5 x 107viable cells) and cryopreserved in a controlled manner to -100.0°C.

[0106] Vials are stored in a vapour phase liquid nitrogen autofill Dewar in a good manufacturing practice (GMP) controlled area.

[0107] Inoculum

[0108] The molecule of the present disclosure is expressed from Chinese Hamster Ovary (CHO) cell lines, in particular the cell line CHOK1SV GS-KO. The cells are grown in medium CM16 (UKSL- 7212).

[0109] Suitable alternative media include:

[0110] CD CHO (Thermofisher)

[0111] CD FortiCHO™ Medium

[0112] CD OptiCHO™ MediumActiCHO (Cytiva)

[0113] EX-CELL® Advanced Medium (Merck Millipore)

[0114] Cellvento CHO (Merck Millipore)

[0115] The inoculum is grown then sequentially transferred via a series of containers of increasing volume: a) 5-30ml b) 30-50ml c) 50-100ml d) 100-200ml e) 200-400ml

[0116] The containers are on a shaker platform and should be maintained at a temperature range of 34- 38C, suitably 36-37C, most suitably at 36.5C. Fermentation

[0117] The grown inoculum material is transferred to 100L cell bag, in CN68 (UKSL-8689) The material should be maintained at a temperature range of 34-38C, suitably 36-37C, most suitably 36.5C. From there it is transferred to a standard air lift type bioreactor.

[0118] The pH should be maintained between 6.7-7.3.

[0119] Antifoam agents as known to those skilled in the art may be used. Suitable agents include:

[0120] Foam away (Thermofisher) & HyClone™ Antifoam (Cytiva)

[0121] A constant O2 flow should be maintained within the reactor, such that the dissolved O2 level in the medium is 25-65%.

[0122] For days 0-7 after transfer to the bioreactor, the pH is suitably 6.84-6.96

[0123] Most suitably pH is 6.9.

[0124] For days 7-harvest, pH is suitably 7.04-7.16

[0125] Most suitably pH is 7.10

[0126] Whilst in the bioreactor, the N2 flow rate is 2.8-4.0L / min, suitably 3.2L / min

[0127] In another embodiment the N2 flow rate is 0.6L / min

[0128] Whilst in the bioreactor, the cells are fed. The feeds are provided at different times and with a variety of constituents: a) Lonza’s SF96 (UKSL-17272) provided continuously b) D-glucose provided variably but continuously. Feed is stimulated if levels drop to <3.0g / L.

[0129] Feed is provided as 400g / L c) Lonza’s SF71 (UKSL-8683). This is added on days 3, 6, 8 and 10 in 1.04Kg doses in 5L d) Lonza’s SF54 (UKSL-5118). This is added on days 3, 6, 8 and 10 in 0.352Kg doses in 5L e) Lonza’s SF72 (UKSL-8710). This is added on days 3, 6, 8 and 10 in 1 kg doses in 10L.

[0130] Suitable alternative feeds include: Efficient feeds (A+ B+ or C+) Gibco; EfficientFeed™ (A+ B+ or C)+ AGT™ Supplement; Cellvento®4 Feed COMP (Merck Millipore); EX-CELL® Advanced CHO Feed 1 (Merck Millipore); ActiCHO feeds A and B (Cytiva)

[0131] These can be added singly or in combination and at different times in the process.

[0132] Harvesting

[0133] Cells are harvested within a) 30 hours of cell culture viability reaching 75% max cell concentration; or b) Maximum of 12 days’ post inoculation.

[0134] The cell medium is cooled to 12-18C, prior to harvest. There is no feed; pH is maintained at 7.25 and N2flow is set to sparge. The pH can be adjusted to and maintained at 7.25 if necessary by use of dissolved CO2

[0135] The supernatant is filtered in a three-part process.

[0136] Stage 1 , 1.1 m2 Millipore Millistak DOHC (6 filters / rack)

[0137] Stage 2, 1.1 m2 Millipore Millistak B1 HC (2 filters / rack)

[0138] Stage 3 10” Millipore Duropore KVGL (0.22Um filter into SOOL Bioprocess container).

[0139] The material is sparged at max air flow rate.

[0140] Filtrate is stored at 5-3C

[0141] After filtration from the cells after fermentation, the mixture is purified. Techniques for purification are well known to those skilled in the art. Suitable techniques would include the following 5 step process:

[0142] 1 Running through a column, which binds the Fc region of the glycosylated protein.

[0143] 2 Low pH hold

[0144] 3 Concentrating the material in a buffer then:

[0145] 4 Running through a first column, from which the product is eluted

[0146] 5 Run through a second ion exchange column using a gradient elution.

[0147] Confirmation that this is the correct glycosylated protein can be obtained by a Sandwich Elisa, which will confirm the presence of the P75 region and the Fc.

[0148] Additional confirmation of the glycosylated protein weight may be obtained by Mass Spectroscopy. Suitable techniques are known to those skilled in the art. Specific identification of the glycan groups can be done by techniques known to those skilled in the art. One such technique would be to reduce the glycosylated protein and alkylate in the presence of urea. The N-glycan’s are released with buffer made up in deuterated water. The release of an N-glycan from an asparagine residue results in deamidation of that residue. This results in a +0.98 Da mass shift when released in water and +2.99 Da mass shift in deuterated water. The samples can then be digested with trypsin or glu-C to generate peptides for the Fc and fusion glycosylated protein glycosylation sites. The proteinase digested samples were subjected to LC-MS analysis.

[0149] Preferred CHO cells for use in the disclosure are CHO1SV GSKO cells. Particularly suitable for use are Lonza’s CHO cells from their GS Xceed™ cell line.

[0150] The osmolality of the stable pharmaceutical formulation in accordance with the invention is 120- 550 mOsm / kg.

[0151] The stable pharmaceutical formulation in accordance with the invention is essentially free from visible (human eye inspection) particles. The sub-visible particles (as measured by light obscuration) should fulfil the following criteria: maximum number of particles > 10pm per vial -> 6000 maximum number of particles > 25pm per vial -> 600

[0152] In a further aspect the present invention provides the use of a formulation for the preparation of a medicament useful for treating a Pain or osteoarthritis in a subject comprising administering the formulation described herein to a subject in an amount effective to treat the said disease or disorder.

[0153] In a further aspect the present invention provides a method of treating Pain or osteoarthritis in a subject comprising administering the formulation described herein to a subject in an amount effective to treat the said disease or disorder.

[0154] Pain may include but is not limited to:

[0155] (a) acute pain and / or spontaneous pain,

[0156] (b) chronic pain and or on-going pain,

[0157] (c) inflammatory pain including any one of arthritic pain, pain resulting from osteoarthritis or rheumatoid arthritis, resulting from inflammatory bowel diseases, psoriasis and eczema

[0158] (d) nociceptive pain,

[0159] (e) neuropathic pain, including painful diabetic neuropathy or pain associated with post-herpetic neuralgia, (f) hyperalgesia,

[0160] (g) allodynia,

[0161] (h) central pain, central post-stroke pain, pain resulting from multiple sclerosis, pain resulting from spinal cord injury, or pain resulting from Parkinson’s disease or epilepsy,

[0162] (i) cancer pain,

[0163] (j) post-operative pain,

[0164] (k) visceral pain, including digestive visceral pain and non-digestive visceral pain, pain due to gastrointestinal (Gl) disorders, pain resulting from functional bowel disorders (FBD), pain resulting from inflammatory bowel diseases (IBD), pain resulting from dysmenorrhea, pelvic pain, cystitis, interstitial cystitis or pancreatitis,

[0165] (I) musculo-skeletal pain, myalgia, fibromyalgia, spondylitis, sero-negative (non-rheumatoid) arthroplasties, non-articular rheumatism, dystrophinopathy, Glycogenolysis, polymyositis, pyomyositis,

[0166] (m) heart or vascular pain, pain due to angina, myocardial infarction, mitral stenosis, pericarditis, Raynaud’s phenomenon, scleroderma, scleroderma or skeletal muscle ischemia,

[0167] (n) head pain including migraine, migraine with aura, migraine without aura cluster headache, tension-type headache.

[0168] (o) orofacial pain, including dental pain, temporomandibular myofascial pain or tinnitus, or

[0169] (p) back pain, bursitis, menstrual pain, migraine, referred pain, trigeminal neuralgia, hypersensitisation, pain resulting from spinal trauma and / or degeneration or stroke.

[0170] Treatment of pain includes, but is not limited to, preventing, ameliorating, controlling, reducing incidence of, or delaying the development or progression of pain and / or a symptom of pain.

[0171] In preferred embodiments, the treatment of osteoarthritis includes relief from the symptoms of osteoarthritis. Suitably relief from the symptoms of osteoarthritis include, but are not limited to reduction in pain, inflammation, swelling, tenderness, joint stiffness or increase in joint mobility or any combination of these.

[0172] In a particularly preferred embodiment, treatment of osteoarthritis includes slowing or arresting of disease progression and / or reduction in cartilage loss. Suitably treatment of osteoarthritis includes reversal of disease progression, regrowth of cartilage and / or curative treatment. Suitably disease progression is determined by the rate of cartilage loss or regrowth. In other preferred embodiments, disease progression may be monitored by determining the number of chondrocytes present in a joint.

[0173] In other preferred embodiments, the treatment of osteoarthritis includes prophylactic treatment.

[0174] The stable pharmaceutical formulation of the present invention can be administered as subcutaneous injection.

[0175] Suitably, subcutaneous administration is to the abdomen.

[0176] The stable pharmaceutical formulation of the present invention can be administered as an intravenous injection.

[0177] The addition of the hyaluronidase to the formulation allows increasing the injection volume which can be safely and comfortably administered subcutaneously.

[0178] For subcutaneous delivery, the formulation may be administered via a suitable device, such as (but not limited to) a syringe; an injection device (e.g. the INJECT-EASE™ and GENJECT™ device); an infusion pump (such as e.g. Accu-Chek™); an injector pen (such as the GENPEN™; an needleless device (e.g. MEDDECTOR™ and BIOJECTOR™); or via a subcutaneous patch delivery system. A suitable delivery system for the formulations in accordance with the present invention is described in WO 2010 / 029054 . Such device comprises about 5 to about 15 ml or more particularly 5 ml of the liquid formulation in accordance with the present invention.

[0179] For the prevention or treatment of disease, the appropriate dosage of the glycosylated protein of the present invention will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, on the previous therapy, the patient's clinical history and their response to the a, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0180] Other therapeutic regimens may be combined with the glycosylated protein of the present invention,

[0181] In another embodiment of the invention, an article of manufacture is provided which contains the pharmaceutical formulation of the present invention and provides instructions for its use. This article of manufacture comprises a container. Suitable containers include, for example, bottles, vials (e.g. multiple or dual chamber vials), syringes (such as multiple or dual chamber syringes) and test tubes. The container may be formed from a variety of materials such as glass or plastic. The container holds the formulation and the label on, or associated with, the container may indicate directions for use. The container holding the formulation may be a multi-use vial, which allows for repeat administrations (e.g. from 2 to 6 administrations) of the reconstituted formulation. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0182] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile.

[0183] A "sterile" formulation is aseptic or free from all living microorganisms and their spores.

[0184] A "stable" formulation is one in which all the protein therein essentially retain their physical stability and / or chemical stability and / or biological activity upon storage at the intended storage temperature, e.g. 2 - 8°C. It is desired that the formulation essentially retains its physical and chemical stability, as well as its biological activity upon storage. The storage period is generally selected based on the intended shelf-life of the formulation. Furthermore, the formulation should be stable following freezing (to, e.g., -70° C) and thawing of the formulation, for example following 1 , 2 or 3 cycles of freezing and thawing. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301 , Vincent Lee Ed., Marcel Dekker, Inc., New York, New York, Pubs. (1991 ) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993 ), for example. Stability can be measured at a selected temperature for a selected time period. Stability can be evaluated qualitatively and / or quantitatively in a variety of different ways, including evaluation of aggregate formation (for example using size exclusion chromatography, by measuring turbidity, and / or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometric analysis; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS-C) analysis; evaluating biological activity or antigen binding function of the antibody; etc. Instability may involve any one or more of: aggregation, deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation), isomerization (e.g. Asp isomeriation), clipping / hydrolysis / fragmentation (e.g. hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation changes, etc. A "deamidated" monoclonal antibody herein is one in which one or more asparagine residue thereof has been modified, e.g. to an aspartic acid or an iso-aspartic acid by a post-translational modification.

[0185] As used herein the term "buffering agent providing a pH of 6.5 ± 2.0" refers to an agent which provides that the solution comprising it resists changes in pH by the action of its acid / base conjugate components. A pH of about 5.5-6.5 has to be found to be most suitable. Examples of buffering agents that will control the pH in this range include acetate, succinate, gluconate, histidine, citrate, glycylglycine and other organic acid buffers. The most suitable buffer in accordance with the present invention is a histidine buffer, such as e.g. L-histidine / HCl. A "histidine buffer" is a buffer comprising the amino acid histidine. Examples of histidine buffers include histidine chloride, histidine acetate, histidine phosphate, histidine sulfate. The histidine buffer identified in the examples as being most suitable is a histidine chloride buffer. Such histidine chloride buffer is prepared by titrating L-histidine (free base, solid) with diluted hydrochloric acid. In particular the histidine buffer or histidine chloride buffer is at pH of 5.5 ± 0.6, more particularly at a pH from about 5.3 to about 5.8, and most particularly has a pH of 5.5.

[0186] By "isotonic" is meant that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing-point depression type osmometer.

[0187] A "saccharide" herein comprises the general composition (CH2 O)n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, nonreducing sugars, etc. Examples of saccharides herein include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, dextran, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, mellibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, iso-maltulose, etc. Particularly the formulations described herein comprise a non-reducing disaccharide as a stabilizing agent, such as a saccharide selected from the group of trehalose (e.g. in the form of a,a-trehalose dihydrate) and sucrose.

[0188] Herein, a "surfactant" refers to a surface-active agent, e.g. a nonionic surfactant. Examples of surfactants herein include polysorbate (for example, polysorbate 20 and, polysorbate 80); poloxamer (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQU AT™ series (Mona Industries, Inc., Paterson, New Jersey); polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc); etc. Polysorbate 20 (PS20) and Polysorbate 80 (PS80), respectively have been found to be particularly suitable in the formulations described herein.

[0189] "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disease as well as those in which the disease is to be prevented. Hence, the patient to be treated herein may have been diagnosed as having the disease or may be predisposed or susceptible to the disease.

[0190] The invention will be more fully understood by reference to the following Examples. They should not, however, be construed as limiting the scope of the invention. All literature and patent citations are incorporated herein by reference. Examples

[0191] The formulations for subcutaneous administration according to the invention were developed based on the experimental results as provided below using the general preparatory and analytical methods and assays as outlined below.

[0192] Liquid drug product formulations for administration according to the invention were developed as follows.

[0193] Tested Formulations:

[0194] Materials used to formulate molecule

[0195] Equipment used during rheological characterization and viscosity screening

[0196] Consumables used during rheological characterization and viscosity screening

[0197] Example 1 preparation of formulations

[0198] For the preparation of the liquid formulations, in the table below, the glycosylated protein of Seq ID No. 1was buffer-exchanged against a diafiltration buffer containing the anticipated buffer composition and, when required, concentrated by diafiltration to the required antibody concentration Example 2: Preparation of a lyophilized formulation

[0199] A solution of approx. 60 mg / ml glycosylated protein of Seq ID No. 1 was prepared as described above for liquid formulations. All excipients have been added at half of the concentration of the above mentioned liquid formulation. The formulation was sterile filtered through 0.22 pm filters and aseptically distributed in equal amounts into sterile 20 ml glass vials. The vials were partly closed with ETFE (Copolymer of ethylene and tetrafluoroethylene)-coated rubber stoppers suitable for the use in lyophilization processes and lyophilized using the freeze-drying cycle reported in below:

[0200] The product was first cooled from room temperature to approx 5°C (pre-cooling), followed by a freezing step at -40°C with a plate cooling rate of approx. 1 °C / min, followed by a holding step at - 40°C for about 2 hours. The first drying step was performed at a plate temperature of approx. - 25°C and a chamber pressure of approx. 80 pbar for about 76 hours. Subsequently, the second drying step started with a temperature ramp of 0.2°C ! min from -25°C to 25°C, followed by a holding step at 25°C for at least 5 hours at a chamber pressure of approx. 80 pbar.

[0201] Lyophilization was carried out in a Usifroid SMH-90 LN2 freeze-dryer (Usifroid, Maurepas, France) or a LyoStar II Freeze-dryer (FTS Systems, Stone Ridge, NY, USA). The freeze-dried samples were stored at different climate conditions (5°C, 25°C and 30°C) for different intervals of time. The lyophilized vials were reconstituted to a final volume of 2.65 ml with water for injection (WFI) yielding an isotonic formulation with an antibody concentration of approx. 120 mg / ml. The reconstitution time of the freeze-dried cakes was around 10 min. Analysis of the reconstituted samples was performed after a 24 hour incubation period of the reconstituted liquid sample at ambient temperature.

[0202] Example 3 -Pharmacokinetic Profile of Protein in the Rat The tested formulation was a 10mg / ML formulation of glycosylated protein comprising Seq ID No.1 additionally comprising 25mM histidine, 50 mM Sodium Chloride, 200 mM Mannitol and with a pH 6.5. (ID F3)

[0203] The protein of Seq ID No. 1 exposure has been determined following IV infusion of the protein of Seq ID No. 1 as part of the regulatory toxicology studies and the data have been used to model the PK profile of the protein of Seq ID No. 1 in the rat. In the non-GLP exploratory toxicology study in rat, a 10-minute IV infusion of the protein of Seq ID No. 1 (3, 15 mg / kg) once a week, for four weeks resulted in a Cmax of the protein of Seq ID No. 1 at the first sampling time, immediately following infusion. Following the fourth administration on Day 22, the the protein of Seq ID No. 1 plasma concentration in animals receiving 15 mg / kg / weekwas 1468 pg.h / mL (area under curve [AUC]o-i68h) and 601 .76 pg / mL (Cmax). On Day 22, the Cmax values were almost double those achieved on Day 1 , indicating accumulation of the compound within the plasma following repeated weekly dosing. A similar toxicokinetic profile of the protein of Seq ID No. 1was seen in the GLP regulatory study. Peak exposure was generally observed 5 minutes after the end of the 10 minute IV infusion, and then remained at a steady level until the end of the sampling period (68 h) post Day 1 dose. Table below provides a summary of parameters determined from the serum concentration of the protein of Seq ID No. 1 measured in the GLP toxicology study in the rat.

[0204] Toxicokinetic parameters of THE PROTEIN OF SEQ ID NO 1 derived from the GLP study in rat

[0205] *tau = 168 h on both sampling days

[0206] Serum concentration of the protein of Seq ID No. 1 versus time profiles were consistent with IV infusion administration - peak exposure was generally observed 5 minutes after the end of the 10 minute IV infusion, followed by a steady concentration or slight increase (at 30 mg / kg in females only) up to the end of the sampling period (68 h) on Day 1 . Following dosing on Day 22 and over the recovery period, plasma concentrations declined gradually up to the end of the sampling period (648 h after last dose was given).

[0207] Overall, the systemic exposure to the protein of Seq ID No. 1 increased in a generally doseproportional manner across the dose range on Days 1 and 22 in both sexes. The Tmax was consistently observed at 0.25 h after the start of the intravenous infusion on Days 1 and 22 in all profiles at each dose level except at 30 mg / kg in males on Day 22 (6.17 h). No trends in T1 / 2, were noted with increasing dose in males and females on both sampling occasions. Following weekly repeat dosing, systemic exposure was generally greater on Day 22 compared with Day 1 at all dose levels in both sexes. No sex-related differences in exposure to the protein of Seq ID No. 1were noted at any dose level on Days 1 and 22.

[0208] Pharmacokinetic parameters for the protein of Seq ID No. 1 estimated from single dose IV, SC and day 1 data from multiple dose IV studies in the rat are seen in Table below. A two- compartment pharmacokinetic model was employed to fit the the protein of Seq ID No. 1 exposure data

[0209] The clearance may be underestimated as some rats in the dataset didn’t show a clear elimination phase. No sex-related differences in exposure to the protein of Seq ID No. 1 were noted.

[0210] Estimated rat PK parameters of the protein of Seq ID no 1

[0211] V1 , central volume; V2, peripheral volume; CL, clearance; Q, inter compartment-clearance; SE, standard error; CV, covariance; Ka, Absorption rate constant; F, Bioavailability

[0212] Plasma concentration following IV administration used to estimate V1 , V2, Q & CL

[0213] Plasma concentration following SC administration used to estimate F and Ka

[0214] Example 4 Stability Screening

[0215] The protein stability in all formulations was assessed in a short-term thermal stability screening.

[0216] The primary packaging materials were prepared as appropriate (washed and sterilized at DPS if not stated otherwise) and each formulation was filled manually applying aseptic handling techniques. Vials were filled manually at a target fill volume of 2.4 ml. All vials were crimped, labelled and put on stability at 25 °C or 40 °C in an upright orientation or tested on the same day for TO.

[0217] The Physico-chemical properties for most of the formulations showed no change, or only moderate change, upon storage at both storage temperatures

[0218] Example 5 - viscosity testing The rheological behaviour of the protein solutions was assessed at 20 °C with a shear-rate ramp using a cone-plate rheometer. The optimal shear-rate to observe Newtonian behaviour was identified at 4000 s-1. This shear rate was therefore used to determine viscosity of prepared protein solutions at 20°C. The measurements at 20 °C were performed in duplicates and the arithmetic mean values were calculated to acquire additional data representative of the manufacturing and injection / administration temperature.

[0219] Viscosity ranged from 15-20 mPa s

[0220] Example 6 -Osmolarity

[0221] The Osmolarity of the formulations was tested and results ranged from 124-510 mOsm / kg.

Claims

Claims1 ) A pharmaceutically acceptable formulation comprising: a) 10-360mg / ml of a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising a p75NTR(NBP) portion, having at least 85% sequence identity with Seq ID No. 2; and an immunoglobulin Fc portion, wherein, the p75NTR(NBP) and Fc portions are connected via a linker, the linker comprises a peptide of formula Gx, where x is 1 , 2, 3, 4, 5 or G.wherein the linker does not comprise or consist of the sequence GGGGS; b) A buffering agent c) A stabilising agent and a pharmaceutically acceptable diluent or carrier.2) The formulation of claim 1 wherein the glycosylated linker portion is GGG and the Fc is a human Fc.3) The formulation of claim 1 or 2 wherein the glycosylated protein has at least 85% sequence identity with the protein of Seq ID No. 1.4) The formulation of claim 1 wherein the glycosylated protein comprises Seq ID No. 1.5) The formulation of claims 1-4 wherein the carrier is saline.6) The formulation of claims 1-5 wherein the formulation is suitable for subcutaneous injection.7) The formulation of claims 1-5 wherein the formulation is suitable for intraveneous injection.8) The formulation of claims 1-7 wherein the stabiliser is a sugar.9) The formulation of claim 8 wherein the sugar is mannitol.10) The formulation of claims 1-9 herein the buffer is a histidine buffer.11) The formulation of claims 1-10 wherein the formulation volume doesn't exceed 2ml.12) A pharmaceutically acceptable formulation comprising a 10mg / ML formulation of glycosylated protein comprising Seq ID No. 1 additionally comprising 25mM histidine, 50 mM Sodium Chloride, 200 mM Mannitol and with a pH 5.9-6.5.

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