Modified bacterial hyaluronidase polypeptides, methods of production, pharmaceutical compositions and uses thereof

Abstract

The present invention relates to modified bacterial hyaluronidase polypeptides, methods for their production, pharmaceutical compositions comprising the modified bacterial hyaluronidase polypeptides of the invention, and uses thereof.

Description

[Technical field]

[0001] The present invention relates to modified bacterial hyaluronidase polypeptides, methods for their production, pharmaceutical compositions comprising the modified bacterial hyaluronidase polypeptides of the invention and uses thereof. [Background technology]

[0002] Hyaluronic acid is an essential component of the extracellular matrix and a quantitatively significant component of the interstitial barrier. Hyaluronidase is a hydrolase that cleaves hyaluronic acid in D-glucuronic acid and N-acetylglucosamine, increasing the permeability of the interstitial matrix. Hyaluronidases are widely distributed in nature. In humans, six different hyaluronidases, HYAL1-4, HYAL-P1 and PH-20, have been identified, with PH-20 believed to exert the strongest biological activity.

[0003] Today, animal-derived bovine or ovine testicular hyaluronidase as well as synthetic hyaluronidase are clinically applied as adjuvants to increase drug bioavailability for the treatment of extravasation or for the management of complications associated with aesthetic injections of hyaluronic acid-based fillers.

[0004] While animal-derived hyaluronidase poses the risk of transmitting animal diseases, such as spongiform encephalopathies, human and bacterial recombinant hyaluronidases exhibit higher purity and reduce pharmaceutical risks.

[0005] To meet clinical needs, it is an object of the present invention to provide hyaluronidase polypeptides suitable for pharmaceutical use, in particular those exhibiting suitable high purity and / or suitable specific activity, suitable stability and solubility, while at the same time exhibiting time- and cost-effective methods of production. Summary of the Invention [Means for solving the problem]

[0006] The aforementioned objects are at least partially solved by the subject matter of the claimed invention. Advantages (preferred embodiments) are set out in the following detailed description and / or the attached drawings as well as in the dependent claims.

[0007] Thus, a first aspect of the invention relates to a modified bacterial hyaluronidase polypeptide comprising, or consisting of, at least 90% sequence identity to SEQ ID NO:1, wherein the hyaluronidase polypeptide comprises a C-terminal HIS tag of SEQ ID NO:7 and an N-terminal Strep tag of SEQ ID NO:5.

[0008] A second aspect of the invention relates to a nucleic acid encoding a modified bacterial hyaluronidase polypeptide of the invention, preferably the nucleic acid comprises or consists of SEQ ID NO:2.

[0009] A third aspect of the invention relates to a recombinant expression vector comprising a vector and a nucleic acid of the invention.

[0010] A fourth aspect of the invention relates to a host cell transformed with a recombinant expression vector of the invention, preferably the host cell is E. coli.

[0011] A fifth aspect of the invention is a method for producing a purified bacterial hyaluronidase polypeptide, comprising the steps of: a. culturing a transformed host cell of the invention in a suitable growth medium under suitable growth conditions to express the bacterial hyaluronidase polypeptide of the invention; b. harvesting the cultured transformed host cells of step a); c. lysing the harvested host cells of step b) and separating the resulting host cell fragments from the resulting host cell contents containing the bacterial hyaluronidase polypeptides of the invention; and d. purifying the resulting host cell content of step c) using HIS affinity chromatography and STREP affinity chromatography to obtain a purified bacterial hyaluronidase polypeptide of the invention; The present invention relates to a method comprising or consisting of the steps of:

[0012] A sixth aspect of the invention relates to a modified bacterial hyaluronidase polypeptide comprising or consisting of at least 90% sequence identity to SEQ ID NO:1, obtained according to the production methods of the invention.

[0013] A seventh aspect of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a modified bacterial hyaluronidase polypeptide of the invention and one or more pharma- ceutically acceptable excipients.

[0014] An eighth aspect of the invention relates to a method of treating a hyaluronan-associated and / or proteoglycan-associated disease or disorder, preferably selected from the group consisting of homozygous familial hypercholesterolemia (xanthomatosis), heterozygous familial hypercholesterolemia and diabetic foot, comprising administering a modified bacterial hyaluronidase polypeptide of the invention, or a pharmaceutical composition of the invention, to a subject in need thereof.

[0015] The inventive aspects of the invention disclosed above may include any possible (sub)combination of the preferred invention embodiments thereof as set out in the dependent claims or as disclosed in the following detailed description and / or the accompanying drawings, as long as the resulting combination of features is reasonable for a person skilled in the art.

[0016] Further features and advantages of the present invention will become apparent from the accompanying drawings. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 depicts a vector map plasmid of a recombinant expression vector of the present invention. [Diagram 2] FIG. 2 depicts a scan of a stained gel after SDS-Page analysis, including various concentrations of a reference protein (bovine serum albumin, syn:BSA) band, a ladder protein band and various concentrations of a modified bacterial hyaluronidase of the present invention band. [Diagram 3] FIG. 3 represents images of LB agar plates over a four day period, including an E. coli positive control (FIG. 3a), an LB plate negative control (FIG. 3b), and the d016 sample of the present invention (FIG. 3c). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] As described in more detail below, the inventors of various aspects of the invention have found that modified bacterial hyaluronidase polypeptides of the invention comprising, or consisting of, at least 90% sequence identity to SEQ ID NO:1 and characterized in comprising a C-terminal HIS tag of SEQ ID NO:7 and an N-terminal Strep tag of SEQ ID NO:5 exhibit high purity of greater than 98.8% (see Examples, section 1.4.1) and high specific activity of 1,500,000 U / mg (see Examples, section 1.4.2).

[0019] In contrast, comparable hyaluronidases, such as bovine hyaluronidase, exhibit a broader range of lower specific activities, i.e., in the range of 300-15,000 U / mg (see Examples, section 2.3). PH20, which is considered to be the most active of the human hyaluronidases, also exhibits lower specific activities, i.e., in the range of 40,000 and 50,000 U / mg. The specific activity of bacterial hyaluronidase from Streptomyces koganeiensis is also comparable to that of PH20, and therefore lower than the specific activity of the modified bacterial hyaluronidases of the present invention.

[0020] Furthermore, the modified bacterial hyaluronidase of the present invention exhibits suitable stability and solubility, as shown in section 1.4.5 of the Examples below. Increased stability, including stability (half-life) against (exo)peptidases, can be attributed to the use of the respective C-terminal HIS tag of SEQ ID NO:7 and the respective N-terminal Strep tag of SEQ ID NO:5. The respective tags can also increase the solubility of the modified bacterial hyaluronidase of the present invention compared to wild-type hyaluronidase (see SEQ ID NO:3, DNA encoding wild-type hyaluronidase see SEQ ID NO:4). Due to the increased stability and solubility, the modified bacterial hyaluronidase of the present invention is preferred for formulating pharmaceutical compositions, particularly parenteral injection compositions.

[0021] Thus, the production methods of the present invention for providing purified modified bacterial hyaluronidase provide relatively high yields and at the same time high purity and high specific activity already on laboratory scale, and are time- and cost-effective in terms of the production process.

[0022] In the context of the present invention, the expression "a modified bacterial hyaluronidase polypeptide of the invention comprising or consisting of at least 90% sequence identity to SEQ ID NO:1, characterized in that it comprises a C-terminal HIS tag of SEQ ID NO:7 and an N-terminal Strep tag of SEQ ID NO:5" is used synonymously with "bacterial hyaluronidase polypeptide of the invention", "bacterial hyaluronidase of the invention" or "hyaluronidase of the invention", and this expression means that the hyaluronidase of the invention comprises at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% of the sequence of SEQ ID NO:1 or consists of 100% of SEQ ID NO:1, and that the hyaluronidase of the invention simultaneously exhibits a C-terminal HIS tag of SEQ ID NO:7 and an N-terminal Strep tag of SEQ ID NO:5. When the sequence of the hyaluronidase of the invention consists of 100% of SEQ ID NO:1, the hyaluronidase of the invention is also synonymously referred to as "d016".

[0023] Thus, the modified bacterial hyaluronidase of the present invention, preferably d016, is suitable for use in pharmaceutical compositions.

[0024] Considering the exhibited relatively high specific activity, purity, stability, solubility and safety profile, the modified bacterial hyaluronidase of the present invention, preferably d016, is suitable for use in the treatment or prevention of hyaluronan-related and / or proteoglycan-related diseases or disorders. The inventors have found that the modified bacterial hyaluronidase of the present invention, preferably d016, is particularly suitable for use in the treatment or prevention of homozygous familial hypercholesterolemia (xanthomatosis), heterozygous familial hypercholesterolemia or diabetic foot.

[0025] Homozygous familial hypercholesterolemia (HoFH) is a rare genetic disease with a prevalence of 1 in 1 million. One form of familial hypercholesterolemia is a lipid metabolism disorder. Patients with HoFH have deposits in the skin and tendons, so-called xanthomas (yellowish spherical lipid deposits), which become noticeable due to a marked increase in the cholesterol fraction in the blood, low-density lipoprotein (LDL). Lipids are also deposited in the walls of blood vessels, causing the early development of severe atherosclerosis with a significant shortening of life expectancy. Patients suffer their first heart attack already from about the age of 5, in early childhood, have to undergo weekly lipid apheresis and take the highest doses of co-medication, but unfortunately their life expectancy is only about 30 years.

[0026] HoFH should be differentiated from the much more frequent heterozygous familial hypercholesterolemia (HeFH), which is also associated with a significantly higher risk of early cardiovascular events, but less dramatically. The prevalence of this genetic disease is estimated at 1:200 to 1:500. Here too, signs of arteriosclerosis, i.e. diseases and symptoms caused by vascular disease, such as peripheral arterial occlusive disease, narrowing of the coronary arteries up to heart attacks, strokes, may develop at a young age, but later and to a lesser extent than in the homozygous variant of the disease.

[0027] The bacterial hyaluronidase of the present invention is effective in the treatment and / or prevention of HoFH and HeFH in view of its reducing effect on tissue extracellular matrix (ECM) and chondroitin-6-sulfate and other proteoglycan levels in atherosclerotic plaques and arteriosclerotic vessel walls. Thus, administration of a therapeutically effective amount of the modified bacterial hyaluronidase of the present invention, preferably d016, results in a reduction in the size of stenotic plaques and an increase in the elasticity of the vessel wall.

[0028] Diabetic foot disease (DFS) is a syndrome of pathological changes based on painless sensory neuropathy and / or peripheral arterial occlusive disease (PAVK) in diabetes. It is most common in type 2 diabetes patients and is associated with a high risk of poorly healing foot wounds. Approximately 15% of diabetic patients will develop painless (due to sensory neuropathy) poorly healing foot wounds during their lifetime. Every year, approximately 4% of diabetic patients will develop new wounds and 0.1% will develop the so-called Charcot foot due to the collapse of the arch.

[0029] Considering the PAVK aspect of diabetic foot, the bacterial hyaluronidase of the present invention is effective in the treatment and / or prevention of diabetic foot by showing its reducing effect on tissue extracellular matrix (ECM) and chondroitin-6-sulfate and other proteoglycan levels in atherosclerotic plaques and arteriosclerotic vascular walls. It thus leads to a reduction in the size of stenotic plaques and an increase in the elasticity of vascular walls. Furthermore, the bacterial hyaluronidase of the present invention has an effect on the neuropathic aspect of diabetic foot. In this regard, it reduces the increased hyaluronic acid concentration present in the nerve sheath area caused by chronic polyneuritis. The associated nerve transmission disorder based on the disturbance of the insulating function of the myelin sheath is reduced and nerve transmission ability is increased again.

[0030] Additionally, the present invention provides, in a second aspect, a nucleic acid encoding the modified bacterial hyaluronidase polypeptide of the invention of the first aspect. All features and embodiments disclosed with respect to the first aspect of the invention can be combined with the second aspect of the invention, including each of its preferred embodiments, alone or in (sub)combination, as long as the resulting combination of features is reasonable to the skilled artisan.

[0031] According to a preferred embodiment of the second aspect of the invention, the nucleic acid comprises or consists of SEQ ID NO: 2. The nucleic acid sequence encoding the C-terminal HIS tag of the hyaluronidase of the invention is preferably SEQ ID NO: 8, and the nucleic acid sequence encoding the N-terminal Strep tag of the hyaluronidase of the invention is preferably SEQ ID NO: 6. The nucleic acids of the invention can be prepared according to any suitable method. Exemplary methods are described in the Examples section 1.1.1 below.

[0032] In the context of the present invention, the expression "nucleic acid of the invention" refers to a nucleic acid encoding a modified bacterial hyaluronidase of the invention, preferably d016. The nucleic acid of the invention preferably comprises or consists of at least 90% sequence identity to SEQ ID NO:2, and preferably the nucleic acid of the invention consists 100% of SEQ ID NO:2.

[0033] According to a third aspect of the invention, a recombinant expression vector comprises the nucleic acid of the second aspect of the invention. All features and embodiments disclosed with respect to the first or second aspects of the invention can be combined with the third aspect of the invention, including each of its preferred embodiments, alone or in (sub)combination, as long as the resulting combination of features is reasonable for the person skilled in the art.

[0034] The recombinant expression vector of the third aspect of the invention can be prepared according to any suitable method. An exemplary method is described in the Examples section 1.1.1 below. The recombinant expression vector of the invention comprises the nucleic acid of the invention comprising or consisting of SEQ ID NO:2, any suitable vector, such as pET-28a DNA vector, in particular pET28a using NcoI / BlpI restriction sites. The recombinant expression vector of the invention can be in any suitable form, such as in the form of a plasmid.

[0035] According to a fourth aspect of the invention there is provided a host cell transformed with a recombinant expression vector according to the third aspect of the invention. All features and embodiments disclosed with respect to the first to third aspects of the invention may be combined with the fourth aspect of the invention, including each of its preferred embodiments, alone or in (sub)combination, as long as the resulting combination of features is reasonable for a person skilled in the art.

[0036] The host cell of the fourth aspect of the invention may be prepared according to any suitable method. Exemplary methods are described in the Examples section 1.1.2 below. The host cell of the invention may be selected from any suitable host cell. Preferably, the host cell is selected from E. coli cells, more preferably E. coli BL21(DE3) competent cells, which provide relatively high yields of the modified bacterial hyaluronidase of the invention, preferably d016.

[0037] The fifth aspect of the invention provides a method for producing a purified bacterial hyaluronidase polypeptide according to the first invention aspect. All features and embodiments disclosed with respect to the first to fourth aspects of the invention may be combined with the fifth aspect of the invention, including each of its preferred embodiments, alone or in (sub)combination, as long as the resulting combination of features is reasonable to the skilled artisan.

[0038] The production method of the present invention can be carried out at laboratory scale using conventional shaker flasks or can be scaled up for use in fermenters. Considering the scale-up to fermenter production, the process steps can be optimized considering high density growth, onset / offset of expression, mixing speed and time, and applicable temperature. The inventors have found that considering the high specific activity of the modified bacterial hyaluronidase of the present invention, preferably d016, the laboratory scale already using shaker flasks is sufficient to produce a significant amount (annual amount) of the modified bacterial hyaluronidase of the present invention, preferably d016, for use in the treatment and / or prevention of homozygous familial hypercholesterolemia (xanthomatosis), heterozygous familial hypercholesterolemia, or diabetic foot.

[0039] The process according to the fifth aspect of the invention comprises or consists of the following steps:

[0040] Step a) culturing the transformed host cell according to the fourth aspect of the present invention in a suitable growth medium under suitable growth conditions for expressing the modified bacterial hyaluronidase polypeptide of the present invention according to the first aspect of the present invention. As an example, Terrific Broth (TB) medium can be used. The respective broth medium may be supplemented with a suitable antibiotic, such as kanamycin, and / or a buffer component, such as potassium phosphate buffer. The growth medium also contains a suitable expression inducer, such as isopropyl β-D-1-thiogalactopyranoside (IPTG). According to the laboratory scale, cell growth and protein expression may be performed in a shaker culture flask at a suitable temperature, preferably 28-30°C, and with a shaking of preferably 180 rpm, for 18-20 hours.

[0041] Step b) harvesting the cultured transformed host cells of step a). After culturing the transformed host cells under suitable growth conditions, the host cells are harvested in a suitable manner. According to laboratory scale, preferably pyrogen-free sterile tubes are used to harvest the harvested host cells containing the growth medium. This harvested growth medium is preferably centrifuged at a low temperature, preferably 4°C, preferably at 4,000rcf, for at least 30 minutes in order to separate the medium from the host cells, which after centrifugation aggregate into a so-called pellet. The supernatant is discarded and the harvest tube is preferably sealed and stored at a low temperature, preferably below 0°C, more preferably at -80°C.

[0042] Step c) Lysing the harvested host cells of step b) and separating the resulting host cell fragments from the resulting host cell contents containing the bacterial hyaluronidase polypeptide of the present invention. According to laboratory scale, the host cells form a flocculated pellet after centrifugation and are generally resuspended and mixed, preferably by vortexing, in a suitable medium, preferably a suitable buffer medium such as phosphate buffered saline (PBS; containing 280 mM NaCl, 6 mM KCl, 15.1 mM Na2HPO4, 4.9 mM NaH2PO4 at room temperature, pH=7.4). The host cells are lysed by any suitable method, such as sonication. To maintain a suitable temperature in the medium, sonication is preferably performed at low temperature, more preferably by surrounding the tube with ice while sonicating the medium. The resulting host cell fragments are separated from the bacterial hyaluronidase polypeptide of the present invention using a suitable method, preferably by centrifugation, more preferably by centrifugation at low temperature, for example at 4°C for at least 30 minutes, for example at 4,000 rcf. The supernatant containing the bacterial hyaluronidase of the invention is preferably transferred to a new tube and optionally subjected to one or more further centrifugation steps. The resulting supernatant containing the bacterial hyaluronidase of the invention is then used for purification step d).

[0043] Step d) purifying the resulting host cell content of step c) using HIS affinity chromatography and STREP affinity chromatography to obtain the purified bacterial hyaluronidase polypeptide of the first aspect of the invention. Preferably, HIS affinity chromatography and STREP affinity chromatography are performed sequentially, and the order is interchangeable, i.e., HIS affinity chromatography purification is performed first, followed by STREP affinity chromatography, or vice versa. In the following, as one exemplary embodiment, sequential purification is described in which HIS affinity chromatography is performed first, and then STREP affinity chromatography is performed. Considering that HIS affinity chromatography columns are generally cheaper than STREP affinity chromatography columns, a high purity yield can be achieved in a more cost-effective manner when HIS affinity chromatography is performed first.

[0044] According to the present invention, any suitable HIS affinity chromatography can be used to bind the C-terminal HIS tag (amino acids 735 to 740 of SEQ ID NO: 1, see also SEQ ID NO: 7) of the modified bacterial hyaluronidase of the present invention. According to laboratory scale, as an example, one or more suitable gravity HIS purification columns are equilibrated with a common PBS downstream buffer. The contents of each tube obtained after step c) are distributed to one or more HIS columns. Optionally, the HIS purification procedure is repeated once, twice or more, preferably twice. The loaded one or more HIS columns are then preferably washed with a suitable washing medium, for example a 10 mM imidazole PBS solution, and then eluted with a suitable elution medium, for example a 150 mM imidazole PBS solution. Generally, the eluates of one or more HIS columns from the same production flask (tube) are combined and preferably diluted to 35 ml with a common downstream buffer PBS. Preferably, the purified eluate sample containing the modified bacterial hyaluronidase of the present invention is then stored at low temperature, preferably on ice, until subsequent STREP purification.

[0045] According to the present invention, any suitable STREP affinity chromatography can be used to bind the N-terminal STREP tag (amino acids 3 to 10 of SEQ ID NO: 1, see also SEQ ID NO: 5) of the modified bacterial hyaluronidase of the present invention. Depending on the laboratory scale, preferably one or more suitable syringe-based STREP affinity chromatography columns can be used. As an example, one or more syringe-based STREP purification steps are performed, more preferably at a flow rate of less than 5 ml / min. The STREP column (5 ml bed volume) is typically washed and equilibrated with 2 x 25 ml of a common downstream buffer, PBS. An appropriate amount of eluate sample obtained from the HIS purification is applied and allowed to flow through the STREP column. The packed STREP column is then preferably washed with an appropriate amount of a common downstream PBS buffer to remove any residual contaminating proteins. By applying an appropriate elution medium, for example a PBS buffer containing 2.5 mM d-desthiobiotin, the protein of interest, i.e. the modified bacterial hyaluronidase of the present invention, is preferably eluted into a fresh, sterile, pyrogen-free tube. The tube is stored at low temperature, for example on ice, or lyophilized to provide a dry, storable product of the purified modified bacterial hyaluronidase of the invention. The STREP column can be reused after appropriate regeneration according to prior art techniques.

[0046] Optionally, the buffer of the eluate can be exchanged by a suitable method, including centrifuging the eluate to aggregate the bacterial hyaluronidase of the invention, discarding the supernatant, and resuspending the aggregated bacterial hyaluronidase of the invention in a different buffer medium, such as Tris HCL NaCl. According to a preferred embodiment, the resuspended bacterial hyaluronidase of the invention is stored at low temperature, for example on ice, for further post-processing, such as polishing.

[0047] According to a preferred embodiment, the purification process of the present invention further comprises one or more suitable polishing steps to remove final impurities of the bacterial hyaluronidase polypeptide of the present invention in step d) and thus increase its purity. According to the laboratory scale, one or more endotoxin removal steps, such as a polymyxin B-based endotoxin removal step; one or more sterile filtration steps, and one or more particle removal steps can be carried out.

[0048] According to the present invention, lab-scale protein yields have resulted in 0.09-0.13 mg / ml after HIS purification and 0.04-0.06 mg / ml after dual HIS / STREP purification and subsequent polishing purification steps (see Examples section 1.3.4 below), which are expected to increase significantly during fermentation-scale production.

[0049] According to a sixth aspect of the present invention, there is provided a modified bacterial hyaluronidase polypeptide of the present invention, preferably consisting of SEQ ID NO: 1, obtainable by a production method according to the fifth aspect of the present invention. All features and embodiments disclosed with respect to the first to fifth aspects of the present invention may be combined with the sixth aspect of the present invention, including each of its preferred embodiments, alone or in (sub)combination, as long as the resulting combination of features is reasonable for the skilled person.

[0050] According to a seventh aspect of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a modified bacterial hyaluronidase polypeptide of the invention according to the first or sixth aspect of the invention, and one or more pharma- ceutically acceptable excipients. All features and embodiments disclosed with respect to the first to sixth aspects of the invention may be combined with the seventh aspect of the invention, including each of its preferred embodiments, alone or in (sub)combination, as long as the resulting combination of features is reasonable to a person skilled in the art.

[0051] Generally, the therapeutically effective amount of the bacterial hyaluronidase of the present invention depends on the therapeutic use of the pharmaceutical composition. According to the present invention, the term "therapeutically active amount" means that the amount of the modified bacterial hyaluronidase polypeptide of the present invention, preferably the amount of the modified bacterial hyaluronidase polypeptide of the present invention consisting of SEQ ID NO: 1, in the pharmaceutical composition or a pharmaceutical unit dose thereof is suitable for the treatment or prevention of a hyaluronan-associated and / or proteoglycan-associated disease or disorder, preferably selected from the group consisting of homozygous familial hypercholesterolemia (xanthomatosis), heterozygous familial hypercholesterolemia or diabetic foot.

[0052] As an example, when the pharmaceutical composition is a parenteral liquid composition for intravenous application, the bacterial hyaluronidase of the present invention may be provided as a concentrate in a vial containing 1 to 10, preferably 2 or 5 mL, at a therapeutically effective amount between 15,000 and 1,500,000 U / mL. According to a preferred embodiment, a unit dose of the pharmaceutical composition of the present invention contains the modified bacterial hyaluronidase polypeptide of the present invention in a concentration range of 200 U / kg / day to 30,000 U / kg / day. To increase the half-life of the bacterial hyaluronidase of the present invention, the administration scheme may preferably include administration of a suitable bolus amount of the hyaluronidase of the present invention to saturate the exoproteinase and / or endoproteinase, followed by administration of a therapeutically effective unit dose amount. Preferably, for intravenous administration, the subsequent unit dose is administered within 1 hour, or within 30 minutes, or within 15 minutes, or within 5 minutes after the bolus administration of the bacterial hyaluronidase of the present invention.

[0053] Notwithstanding the above, the pharmaceutical composition of the present invention can be in any suitable application form, such as a solid, semi-solid or liquid application form. The therapeutically effective amount should be calculated accordingly. As an example, the solid form of the pharmaceutical composition of the present invention can be provided as a dry or lyophilized form. In addition to the bacterial hyaluronidase polypeptide of the present invention, one or more pharma- ceutically acceptable excipients, such as one or more components selected from the group consisting of bulking agents, buffers, isotonicity adjusters, collapse temperature adjusters, solvents and / or cosolvents, solubilizers, preservatives, antioxidants, antibacterial agents and chelating agents, wetting agents, flocculating / suspending agents, and optionally proteinase inhibitors, such as one or more selected from metalloproteinase inhibitors, dipeptidyl-4 exopeptidase inhibitors (syn:DPP-4 inhibitors or gliptins), or hyaluronan-binding protein 2 protease inhibitors.

[0054] According to the present invention, suitable bulking agents may include sucrose, lactose, trehalose, mannitol, sorbitol, glucose, raffinose, glycine, histidine or polyvinylpyrrolidone (K40). Suitable buffering agents may include sodium citrate, sodium phosphate, sodium hydroxide, Tris base 65, Tris acetate, or Tris HCl 65. Suitable isotonicity adjusting agents may include dextrose. Suitable collapse temperature adjusting agents may be dextran, ficoll, gelatin, hydroxyethyl starch. Suitable solvents are preferably selected from water for injection, and non-aqueous water-miscible agents such as ethanol, glycerin, propylene glycol and n-lactamide may be used as co-solvents. Suitable solubilizing agents may be selected from suitable surfactants and co-solvents. A few examples of suitable surfactants are polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monooleate, polyoxyethylene sorbitan monolaurate (Tween 20), lecithin, polyoxyethylene polyoxypropylene copolymer (Pluronics). Examples of suitable cosolvents as solubilizers are propylene glycol, glycerin, ethanol, polyethylene glycol (300 and 400), sorbitol, dimethylacetamide and Cremophor EL. Suitable preservatives may be selected from parabens, such as benzyl alcohol (0.9% to 1.5%), methylparaben (0.18% to 0.2%), propylparaben (0.02%), benzalkonium chloride (0.01% to 0.02%) and thiomersal (0.001% to 0.01%). Suitable antioxidants are preferably selected from ascorbic acid, sulfites such as sodium bisulfite, sodium metabisulfite, sodium formaldehyde sulfoxylate, thiourea, acetylcysteine, ascorbic acid esters, butylated hydroxytoluene, tocopherol. Suitable antibacterial agents are selected from phenol, meta-cresol, benzyl alcohol, parabens (methyl, propyl, butyl), benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric salts (acetate, borate, nitrate). Suitable chelating agents are selected from ethylenediaminetetraacetate.Suitable wetting agents are preferably selected from glycerin, alcohol and propylene glycol. Suitable flocculating / suspending agents are selected from electrolytes such as potassium / sodium chloride, potassium / sodium citrate or potassium / sodium acetate, or surfactants and hydrophilic colloids such as sodium carboxymethylcellulose, acacia, gelatin, methylcellulose, polyvinylpyrrolidone. Proteinase inhibitors can extend the half-life of the bacterial hyaluronidase of the present invention, thus reducing the total amount of hyaluronidase used or improving the therapeutic efficacy. Calcium disodium edetate can be used to broadly inhibit metalloproteases. Vildagliptin or linagliptin can be used to specifically inhibit DPP-4 exopeptidase and can cleave sequences with proline. Aproptin may be suitable for inhibiting hyaluronan binding protein 2 (HABP2) and may be suitable for inhibiting a wide range of serine proteases.

[0055] In the case of a lyophilized pharmaceutical composition, one or more excipients may be selected from suitable bulking agents, buffers, isotonicity adjusters, collapse temperature adjusters, and proteinase inhibitors. In the case of the pharmaceutical composition of the present invention being applied as a parenteral injection, one or more pharmaceutical excipients may be selected from solvents, solubilizers, cosolvents, preservatives, wetting agents / surfactants, flocculation / suspending agents, and proteinase inhibitors.

[0056] Alternatively, each of the one or more proteinase inhibitors may be administered separately into a pharmaceutical composition of the invention that includes a bacterial hyaluronidase of the invention. When administered separately, the one or more suitable proteinase inhibitors are generally administered prior to or simultaneously with the bacterial hyaluronidase of the invention in an amount suitable to effectively inhibit exopeptidases and / or endopeptidases.

[0057] When a pharmaceutical composition of the invention comprising a hyaluronidase of the invention is administered in the form of a bolus dose followed by a subsequent unit dose administration, a protease inhibitor may be included in the bolus and optionally subsequent unit doses of the pharmaceutical composition of the invention. To reduce the body burden of the proteinase inhibitor, preferably only the bolus dose contains the appropriate one or more protease inhibitors, or a separate dose of one or more protease inhibitors is administered prior to or simultaneously with the bolus dose of the pharmaceutical composition of the invention comprising a hyaluronidase of the invention.

[0058] As an alternative example, the liquid form of the pharmaceutical composition of the present invention may be provided as a suspension of the bacterial hyaluronidase polypeptide of the present invention in a suitable suspension medium, preferably in a suitable suspension medium for parenteral application, more preferably in a suitable suspension medium for intravenous application. The pharmaceutical composition may be in the form of a concentrate that is diluted before parenteral application. The pharmaceutical composition for parenteral injection may contain one or more excipients selected from the group of solubilizers, cosolvents, preservatives, wetting agents / surfactants, flocculation / suspending agents and proteinase inhibitors.

[0059] As an example, liquid forms (after dilution) of a pharmaceutical composition of the invention for parenteral injection include 0.9% NaCl solution, Ringer's solution, or sodium lactate-sodium chloride solution.

[0060] In general, the pharmaceutical compositions of the present invention may be suitable for oral, nasal, transdermal, rectal, intravenous or intramuscular application. For oral application, the bacterial hyaluronidase polypeptides of the present invention are preferably formulated with a suitable enteric coating to avoid / reduce the degradation of the bacterial hyaluronidase of the present invention in intestinal fluids. Intravenous application of the pharmaceutical compositions of the present invention is particularly preferred since the bacterial hyaluronidase polypeptides of the present invention are provided directly to the vascular space without the first-pass effect, which is particularly preferred for use in the treatment or prevention of hyaluronan-related and / or proteoglycan-related diseases or disorders, preferably selected from the group consisting of homozygous familial hypercholesterolemia (xanthomatosis), heterozygous familial hypercholesterolemia and diabetic foot. In this case, exopeptidases and endopeptidases can be inhibited by the use of bolus application of the bacterial hyaluronidase of the present invention and / or the further administration of a proteinase inhibitor in the same or another pharmaceutical composition.

[0061] A method for treating a hyaluronan-associated and / or proteoglycan-associated disease or disorder, preferably selected from the group consisting of homozygous familial hypercholesterolemia (xanthomatosis), heterozygous familial hypercholesterolemia and diabetic foot, comprises or consists of administering a modified bacterial hyaluronidase polypeptide of the invention according to the first or sixth aspect, or a pharmaceutical composition of the invention according to the seventh aspect, to a subject in need thereof.

[0062] The present invention will be described below based on exemplary embodiments, which serve only as an example and do not limit the scope of the present protection. EXAMPLES

[0063] Further features and advantages of the present invention will become apparent from the following description of exemplary embodiments of aspects of the invention, which description proceeds with reference to the accompanying drawings.

[0064] All of the features disclosed below with respect to the exemplary embodiments and / or the accompanying drawings may be combined with the features of the aspects of the invention, including the features of its preferred embodiments, alone or in any subcombination, so long as the resulting combination of features is reasonable for a person skilled in the art.

[0065] Equipment (general) 1.Thermo Scientific Multifuge X3R(SN:42343259) 2.Eppendorf Centrifuge 5920R(SN:5948HR902433) 3.New Brunswick Scientific Innova 4300(SN:590544115) 4.New Brunswick Scientific Innova 4300(SN:791060864) 5.Jenway 6305 Spectrophotometer (SN:68993) 6.ARCTIKO ULTF 420-80℃ Freezer (SN:20180262153) 7. Liebherr GX 823-20M Freeza (SN:50.636.690.7) 8.Sartorius Arium Mini UV Ultrapure Water(SN:36802288) 9. VWR Vapour Line 135-B Autoclave (MN:12175020) 10. Peqlab TS-100 Thermoshaker (SN: 430805037) with 24x2ml block (SN: 191165) 11.IKA Vortex 2 S000(SN:100451198) 12.Fisher Scientific Mini300V Plus Power Supply (SN:190118120) 13.IKA Rocker 2D Basic Shaker (SN:100574254) 14.Kern precision balance ABJ320-4NM (SN:WB18AM0063) 15.Kern PBS4200-2M balance (SN:WB17M0023) 16.AQUALYTIC pH-Meter SD AL 10 PH(SN:AJ.12842) 17. Eppendorf 500-5000μl Pipette (SN: G44139 I) 18. Eppendorf 100-1000μl pipette (SN:J37828B) 19. Eppendorf 100-1000μl pipette (SN:H41815D) 20. Eppendorf 10-100μl pipette (SN:H39057D) 21. Eppendorf 10-100μl pipette (SN:H39145D) 22. Eppendorf 0.1-10μl pipette (SN:400555A) 23. Bandelin Sonoplus GM 2200.2 generator with converter UW 2200 (SN: 599.00125433.006) and KE76 horn (SN: 3714.00125432.005) 24. Epson Workforce Pro WF-4720 (Model C582A) (SN: *X2TU046376*) 25.Thermo Scientific NanoDrop Lite(SN:5149)

[0066] Materials / Chemical Substances (General) 1. Glycerin (CAS: 56-81-5) 2. Agar (CAS:9002-18-0) 3. Yeast extract (CAS:8013-01-2) 4. Isopropyl β-D-1-thiogalactopyranoside IPTG (CAS: 367-93-1) 5. Vegetable peptone (CAS: 91079-46-8) 6. Sodium chloride (NaCl) (CAS: 7647-14-5) 7. Potassium chloride KCl (CAS: 7447-40-7) 8. Kanamycin solution (CAS: 25389-94-0) 9. Sodium phosphate monobasic NaH2PO4 (CAS: 13472-35-0) 10. Sodium phosphate dibasic Na2HPO4 (CAS: 7558-79-4) 11. Potassium phosphate monobasic KH2PO4 (CAS: 7778-77-0) 12. Potassium phosphate dibasic K2HPO4 (CAS: 7758-11-4) 13. Imidazole (CAS: 288-32-4) 14. d-Desthiobiotin (CAS: 533-48-2) 15. Hydrochloric acid HCl (CAS: 7647-01-0) 16. Sodium hydroxide (NaOH) (CAS: 1310-73-2) 17. Tris base (CAS: 77-86-1) 18.His GraviTrap TALON(Sigma-Aldrich GE29-0005-94) 19.StrepTrap(TM) High Performance (Sigma-Aldrich GE28-9075-47) 20. Bovine serum albumin (BSA) (CAS: 9048-46-8) 21. Hyaluronic acid (CAS: 9067-32-7) 22. Sodium acetate NaOAc (CAS: 6131-90-4) 23.2x LAEMMLI buffer (β-mercaptoethanol CAS: 60-24-2, sodium dodecyl sulfate CAS: 151-21-3) 24. Tris-MOPS SDS electrophoresis buffer (sodium dodecyl sulfate CAS: 151-21-3, Tris CAS: 77-86-1, EDTA CAS: 60-00-4, MOPS CAS: 1132-61-2) 25. Methanol MeOH (CAS: 67-56-1) 26. Comassie Brilliant Blue R-250 (CAS: 6104-59-2) 27. Bovine hyaluronidase Type IS (CAS:37326-33-3)

[0067] 1: Production of the modified bacterial hyaluronidase polypeptide of the invention of SEQ ID NO:1 1.1 Preparation 1.1.1 Gene synthesis and subcloning - pET28a(+) The d016 gene sequence of SEQ ID NO:2 encoding the modified bacterial hyaluronidase of the present invention of SEQ ID NO:1 was designed by truncating the wild-type sequence of Streptococcus pneumoniae of SEQ ID NO:4 and adding a C-terminal HIS tag gene sequence of SEQ ID NO:8 and an N-terminal Strep tag gene sequence of SEQ ID NO:6 for two-step affinity chromatography purification. By design, both tags were designated to remain on the final enzyme product to increase solubility and protect against (exo)peptidases in the vascular space, the designated drug compartment. Synthesis of the gene construct, cloning into pET28a using NcoI / BlpI restriction sites results in a recombinant expression vector (syn.:plasmid containing pET28a(+) vector with d016 insert). The vector map plasmid of pET28a(+) and insert d016 is shown in FIG. 1.

[0068] 1.1.2 Plasmid transformation into E. coli BL21(DE3) The plasmids were transformed into E. coli BL21(DE3) competent host cells by heat shock at 42° C. according to the supplier's protocol (New England Biolabs). Selection of positive transformants of the host cells of the invention was performed on lysogeny broth (LB) agar plates containing 50 μg / ml kanamycin antibiotic according to the pET28a encoded resistance. Plates were grown at room temperature for 48 hours.

[0069] 1.1.3 Transformation culture of the host cell of the invention in lysogeny broth (LB medium and glycerol stock preparation (stored at -80°C) A single clone of the host cell of the present invention was picked from the plate and cultured overnight at 37°C in LB medium containing 50 μg / ml kanamycin. Sterile pyrogen-free tubes were prepared with sterile 50% glycerol solution. Under sterile laminar flow conditions, the liquid culture grown overnight (12-14 hours) was diluted into a master clone storage tube containing 50% of the liquid culture volume and 50% of the prepared glycerol solution. The master clone was stored at -80°C. All batches of the host cell culture of the present invention in d016 were derived from this master clone.

[0070] 1.2 Manufacturing 1.2.1 Preculture inoculation in LB medium and overnight growth (37°C) Two 250 ml culture flasks (autoclaved) were prepared with (vegetable) LB medium (autoclaved). 50 μg / ml kanamycin was added to prevent the growth of potential bacterial contaminants. Preculture preparation / handling was always performed under sterile laminar flow conditions. One flask containing 65 ml of kanamycin-containing (vegetable) LB medium was inoculated by a single pipette tip (autoclaved) with a sample from the master clone storage tube. The second flask was used as a negative control containing a pipette tip without cells of the master clone. The preculture tube was sealed by a breathable membrane under laminar flow and delivered to an incubation shaker. The cells were grown overnight (12-14 h) at 37 °C and 180 rpm. The master clone storage tube was sealed throughout the entire process and was only opened under laminar flow conditions. After inoculation, the master clone tube was sealed and stored at -80 °C.

[0071] 1.2.2 Inoculate the main culture into Terrific Broth (TB) medium and incubate overnight (28-30 °C) with isopropyl β-D-1-thiogalactopyranoside (IPTG) induced expression Fourteen 500 ml baffled flasks with breathable screw caps were autoclaved and each filled with 200 ml (vegetable) TB medium (autoclaved, vegetable peptone 12 g / l, yeast extract 24 g / l, glycerol 8 ml / l) containing potassium phosphate buffer (individually autoclaved, final concentrations 9.4 g / l K2HPO4 and 2.2 g / l KH2PO4). 50 μg / ml kanamycin was added to prevent the growth of potential bacterial contaminants. Twelve flasks were inoculated individually with 4 ml of preculture using a 5 ml pipette tip (autoclaved). The remaining two flasks containing 4 ml of control preculture were used as controls. Preparation of the main cultures was always performed under sterile laminar flow conditions. All flask caps were screwed tightly before transferring the flasks to an incubation shaker with growth conditions set at 37 °C and 180 rpm. After 3-4 h and a growth OD600 of 0.7-1.1, all flasks were equilibrated to room temperature. Protein production was induced with 200 μl of 100 mM IPTG stock solution. Finally, all flasks (including the contamination control) were placed in a second incubator shaker at 28-30 °C (180 rpm) for 18-20 h.

[0072] 1.2.3 Culture harvest and cell pellet storage (-80°C) After the prescribed growth period of 18-20 hours, the culture flasks were removed from the incubation shaker. The control flasks were checked to ensure that no contaminant growth had occurred within the batch. The controls were not processed further. All 12 culture flasks were harvested using pyrogen-free sterile 50 ml tubes. 3 x 50 ml of each flask was harvested and the remaining volume was discarded. All tubes were centrifuged at 4000 rcf, 4°C for 30-45 minutes. The supernatant was discarded and all tubes containing pellets with the modified bacterial hyaluronidase of the present invention were sealed and stored at -80°C for use in downstream processing.

[0073] 1.3 Downstream Processing 1.3.1 Harvesting / pretreatment: Cell lysis by sonication, fragment removal by centrifugation The pellet was resuspended by vortexing in 10 ml of common downstream buffer PBS (phosphate buffered saline, containing 280 mM NaCl, 6 mM KCl, 15.1 mM Na2HPO4, 4.9 mM NaH2PO4 at room temperature, pH=7.4). Three tubes from the same flask origin were combined for sonication (36 tubes were combined into 12 tubes) and stored on ice until processing. The sonicator was set at 60% amplitude, 2 seconds on, 4 seconds off cycle. Each tube was sonicated individually and surrounded by ice water in a 100 ml glass bottle to ensure a constant low temperature for all samples. After sonication of all samples, the tubes were centrifuged at 4000rcf and 4°C for 45 minutes. The supernatant was transferred to a new sterile pyrogen-free 50 ml tube, making sure that no significant amount of cell fragment material was transferred. An additional round of centrifugation at 4000rcf and 4°C for 45 minutes was performed to remove residual cell fragments. The resulting supernatant containing the modified bacterial hyaluronidase of the present invention was used directly for HIS purification.

[0074] 1.3.2 Purification I: HIS affinity chromatography followed by short storage of the eluate (on ice) Eighteen gravity HIS purification columns were equilibrated with common PBS downstream buffer. The contents of each centrifuge tube were distributed to three HIS columns. The purification procedure was repeated twice for a single batch (2 x 18 columns per batch = 36 column purifications). Untreated tubes were always kept on ice. From each tube, 3 x 9 ml of supernatant sample was carefully transferred to three independent HIS columns by pipetting, so as not to disturb the residual cell pellet remaining in the tube. The packed columns were then washed with 10 ml of 10 mM imidazole PBS solution, followed by elution with 6 ml of 150 mM imidazole PBS solution prepared in a new sterile pyrogen-free tube. The eluates of the three columns originating from the same production flask were combined (a total of 36 purified eluates were combined into 12 tubes) and diluted to 35 ml with common downstream buffer PBS. The purified samples of the modified bacterial hyaluronidase of the present invention were kept on ice until the following STREP purification. The HIS purification column was washed by applying 12 ml of 300 mM imidazole in PBS and prepared for the second run with 12 ml of ultrapure water followed by 12 ml of common downstream buffer PBS.

[0075] 1.3.3 Purification II: STREP affinity chromatography, buffer exchange (TrisHCl NaCl), short-term storage of eluate (on ice) All tubes containing the HIS-purified protein samples of the modified bacterial hyaluronidase of the present invention were stored on ice until the entire STREP purification process was completed. For each of the combined eluate samples from the HIS purification, two replicates of the syringe-based STREP purification step with a flow rate of less than 5 ml / min were performed for each process (12 combined tubes from the HIS purification were processed through 24 STREP columns). The STREP column (5 ml bed volume) was washed and equilibrated with 2 x 25 ml of common downstream buffer PBS. 17.5 ml of eluate sample was applied and passed through the column. The packed column was then washed with 50 ml of common downstream PBS buffer to remove any residual contaminating proteins. The protein of interest was eluted into a fresh sterile pyrogen-free tube by applying 20 ml of 2.5 mM d-desthiobiotin-containing PBS buffer, which was always stored on ice. The column was regenerated with 15 ml of ultrapure water, followed by 15 ml of 0.5M NaOH, followed by 15 ml of ultrapure water, and finally 25 ml of common downstream PBS buffer. At this stage, the next sample was similarly loaded and purified. A single STREP column was used for 12 purification runs, i.e., half of the batch. After purification of the entire batch, buffer exchange was performed via a Sartorius VivaSpin 20 50 kDa centrifugal concentrator. Each VivaSpin was loaded with 20 ml of STREP eluate. Samples were concentrated to less than 1 ml by centrifugation at 4000 rcf and 4°C for 45 minutes. Purified protein of the modified bacterial hyaluronidase of the present invention was collected in VivaSpins and buffer exchanged by adding TrisHCl NaCl (50 mM Tris, 154 mM NaCl, pH=7.4 at room temperature) to 20 ml. This procedure of centrifugal concentration and redilution to 20 ml with Tris HCl NaCl was performed three times from that point on to meet the CoA specifications. After the final centrifugation, the VivaSpin was filled to 5 ml instead of 20 ml. Each VivaSpin was then carefully resuspended and 5 ml was removed into a pyrogen-free sterile tube. Two further resuspensions with 5 ml of Tris HCl NaCl were performed to ensure good recovery of the desired protein from the VivaSpins.All 12 tubes, each containing 15 ml buffer exchanged, of the modified bacterial hyaluronidase protein of the present invention were kept on ice for the polishing step.

[0076] 1.3.4 Optional polishing: endotoxin (LPS removal), particle removal (filtration), sterile filtration, long-term storage (-15°C) For polishing, two polymyxin B-based endotoxin removal steps, two sterile filtration steps and one particle removal step were performed. In preparation, all buffer-exchanged proteins were passed through a sterile pyrogen-free 0.22 μm PES filter. The filtrate was stored on ice in a fresh sterile pyrogen-free tube. The following steps were performed under sterile laminar flow conditions. For each sample, two consecutive endotoxin removal steps on (two separate) immobilized polymyxin B columns (supplied by GenScript) were performed. Each column was washed with a total of 15 ml of the supplied regeneration buffer to remove residual endotoxins, followed by washing with a total of 18 ml of the supplied equilibration buffer (phosphate-based) to remove all of the regeneration buffer. In addition, a total of 15 ml of Tris-HCl NaCl was applied to further remove the phosphate buffer. 15 ml of sterile filtered sample was loaded onto the first column and collected in a new sterile pyrogen-free tube. To collect the remaining protein and increase the yield of the modified bacterial hyaluronidase of the present invention, 5 ml of TrisHCl / NaCl was applied to the column and the flow-through was collected in the same tube. The process was repeated using a fresh second column. Both endotoxin removal columns were briefly regenerated multiple times during the batch run with a total of 10 ml of regeneration buffer, followed by a total of 10 ml of equilibration buffer, and finally followed by a total of 15 ml of TrisHCl NaCl. A total of two sets of endotoxin removal columns were used (for all batches). The endotoxin removal columns were regenerated after three sample runs. These endotoxin removal procedures were performed for all samples and the resulting samples were stored on ice. The samples were then collected in fresh sterile pyrogen-free tubes and transferred to a fresh Sartorius VivaSpin20 100 kDa centrifugal concentrator under laminar flow for centrifugation at 3000 rcf for 20 minutes at 10°C. Under laminar flow conditions, the VivaSpin flow-through was resuspended and collected in a fresh sterile pyrogen-free tube. All flow-through was collected (separately for each sample) and then subjected to another round of sterile filtration through a sterile pyrogen-free 0.22 μm PES filter inside the laminar flow.Using pyrogen-free pipette tips, sample aliquots were made to measure the concentration of protein in the final samples by 280 nm absorbance. Based on this concentration data, the combined product was diluted with Tris HCl NaCl to a final concentration of about 0.1 mg / ml. Small aliquots were prepared from the final product of the batch for batch analysis in sterile pyrogen-free tubes. The final product containing the modified bacterial hyaluronidase d016 of the present invention and all aliquoted batch analysis samples were tightly screwed and sealed with parafilm and stored at -15°C.

[0077] Protein yield [mg / ml] from E. coli BL21(DE3) / pET28a(+) shaker flask expression was determined by measuring the amount of protein [mg] after HIS and STREP purification via absorbance at 280 nm (calculated absorption coefficient of 132590 mol-1 cm-1 and calculated mass of 84516 Da) based on the culture volume [ml] during protein expression.

[0078] Therefore, the yield obtained was approximately 0.09-0.13 mg / ml (after HIS purification). The final yield after HIS purification, STREP purification and polishing was approximately 0.04-0.06 mg / ml.

[0079] 1.4 Batch Analysis 1.4.1 Protein Purity: SDS-PAGE Analysis of Final Batch Purity with Comassie R-250 Staining For analysis of protein purity, a single batch analytical aliquot was thawed and the concentration was confirmed by absorbance at 280 nm. Based on this data, samples with concentrations from 0.1 μg / 25 μl to 16 μg / 25 μl were made to analyze purity by SDS-PAGE. 25 μl of 2x LAMMELI buffer was added to each dilution and mixed gently by pipetting. Samples were then denatured by incubation at 60 °C for 20 min before loading to improve uniform transmission through the gel matrix. This was followed by centrifugation at 20 °C and 6000 rcf for 1 min to return condensate to the sample volume. A GenScript GelBox was filled halfway with GenScript Tris-MOPS SDS running buffer and a ready-to-use SurePage gel was introduced after removal of the safety strip. The inner space was then filled completely with Tris-MOPS SDS running buffer and the comb was gently removed. Individual loading chambers (12 per gel) were washed by pipetting 100 μl Tris-MOPS SDS multiple times to remove glycerol. All denatured and prepared samples (50 μl) were loaded (11 per gel) and an additional chamber was used to apply 5 μl of NEB Prestained Protein ladder. The GelBox was sealed and connected to the power supply (120 V) until the smallest band of the ladder approached the exhaustion of the gel (60–90 min). The power was turned off and the gel was removed from the chamber. After opening the gel cassette, the gel was placed in a staining box filled to the 1 cm level with staining solution (50% MeOH, 40% ultrapure water, 10% acetic acid, 1.0 g / l Comassie R-250). The gel was stained overnight on a rocker shaker. The staining solution was removed the following morning.

[0080] The gel was washed with demineralized water and heated in a microwave to an estimated 70-90 °C. The gel was then transferred to a shaker for 20 min. The process was repeated until bands were clearly visible and background signal was significantly reduced. As a final step, a destaining scan (1200 dpi) was performed (see Figure 2) to assess purity based on the limit of detection by monitoring the visibility of sample contaminating bands in samples from 0.1 µg to 16 µg per lane. The limit of detection was verified by known amounts (0.2 µg and 0.1 µg) of a reference protein (BSA).

[0081] 2 depicts a scan of a test batch containing BSA protein at concentrations of 0.2 μg and 0.1 μg, a ladder reference band, and modified bacterial hyaluronidase of the invention at concentrations of 16 μg, 8 μg, 4 μg, and 2 μg. The modified bacterial hyaluronidase of the invention protein migrates between the 80 kDa and 100 kDa ladder reference bands, confirming that the protein of the invention is the correct size.

[0082] No contaminating bands were detectable at any loading concentration of the modified bacterial hyaluronidase of the present invention (0.1-16 μg), indicating a theoretical purity of the single contaminating protein of greater than 98.8%. This finding is based on a detection limit of 0.2 μg per lane, although the analyzed proteins and BSA can also be detected already at 0.1 μg per lane.

[0083] material: GenScript GenBox Mini Electrophoresis Tank (L00780) NEB Prestained Protein Ladder(P7712) GenScript SurePage Gel 10x8 12% (M00668) Peqlab TS-100 Thermoshaker (see Equipment) Fisher Scientific Mini300V Plus Power Supply (see Equipment) Thermo Fisher Scientific NanoDrop lite (see Equipment) GenScript Tris-MOPS SDS Electrophoresis Buffer (M00138) Sigma-Aldrich (GE) BSA (05470) Sigma-Aldrich LAEMMLI 2x buffer (S3401) Eppendorf pipette (see Equipment) Sigma-Aldrich (Nalgene) staining box (Z358290) Carl-Roth Methanol (KK39.2) Sigma-Aldrich Comassie Brilliant Blue R-250 (27816-25G) Epson Workforce Pro WF-4720 (see equipment) Sartorius Arium Mini Plus (see device) IKA Rocker 2D Basic Shaker

[0084] 1.4.2 Protein activity: specific activity measurements According to the US Pharmacopoeia (USP), the unit activity of hyaluronidase is determined by calibration with the USP National Formulary Reference Standards. The unit activity is defined as follows: "One unit is based on the change in absorbance at 600 nm (change in turbidity) of USP Reference Standard Hyaluronidase assayed simultaneously with each lot of this product."

[0085] Since this standard is no longer available for purchase, the supplier Sigma-Aldrich derived a method to use a previously calibrated reference enzyme. The new unit definition is as follows: "One unit causes a change in A600nm of 0.330 per minute at 37°C and pH 5.7 (45 minute assay)."

[0086] Further: "A change in absorbance value of 0.330 was selected in the new unit definition to most closely match results found using the activity defined in the USP Hyaluronidase standard. As a result, the discontinued USP-based unit definition and the new unit, Sigma-Aldrich unit definition, exhibit a conversion factor of approximately 1:1 (one old unit is approximately equal to one new unit)." Sources for activity unit definitions are also derivable under https: / / www.sigmaaldrich.com / life-science / biochemicals / biochemical-products.html?TablePage=111679355.

[0087] The turbidity assay to measure the activity units of the modified bacterial hyaluronidase of the present invention was performed according to the Sigma Aldrich protocol, including identical preparation of all assay buffers, reaction parameters and measurement procedures. Any modified procedures are described below.

[0088] The USP-based specific activity assay described above was also further calibrated against a "reference" hyaluronidase from Sigma-Aldrich to remove deviations arising from individual measurements. This reference enzyme (Sigma-Aldrich bovine hyaluronidase Type IS [H3506]) was used at various concentrations to generate a standard curve (linear fit) to convert the measured transmittance (blank A600-sample A600) to U / mg. Sigma-Aldrich determined the specific activity according to the protocol described above.

[0089] Standards (3.16U / 1.5ml reaction-6.55U / 1.5ml reaction) were prepared by the dilution procedure. To measure specific activity, each sample of the modified bacterial hyaluronidase of the present invention (syn:d016) was diluted (as needed) in TrisHCl NaCl to a concentration of 0.1mg / ml. Concentrations were confirmed by A280 measurement and the identified deviations were used to correct the corresponding specific activity measurements.

[0090] The sample was then diluted 400-fold in Sigma-Aldrich assay enzyme dilution buffer and 11.7 μl of this diluted sample was used per 1.5 ml reaction to yield 2.93 ng of d016 per 1.5 ml reaction (see Table 1 below). The dilution procedure for 2.93 ng of enzyme was empirically determined to be within the standard absorbance range listed above.

[0091] [Table 1]

[0092] Therefore, the amount of modified bacterial hyaluronidase d016 of the present invention in the final reaction (11.7 μl used per reaction) corresponds to 2.93 ng / reaction.

[0093] For absorbance measurements, 250 μl of the 45 min reaction was combined with 1.25 ml of precipitation buffer (pH=3.75) and measured in a spectrophotometer for A. This value was used to calculate the transmittance and values ​​were converted to specific activity according to the protocol previously described.

[0094] Details of the turbidity method can be found on the Sigma Aldrich website at: https: / / www.sigmaaldrich.com / content / dam / sigma-aldrich / docs / Sigma / General_Information / 2 / hyaluronidase.pdf

[0095] Relative to Sigma-Aldrich bovine hyaluronidase as determined according to USP unit definitions, the modified bacterial hyaluronidase d016S of the present invention provided the respective specific activities as shown in Table 2 below:

[0096] [Table 2]

[0097] Thus, the typical specific activity of the modified bacterial hyaluronidase of the present invention is in the range of 1.5 Mio.USP U / mg + / - 150k USP U / mg, which is equivalent to 25,000 katals / kg + / - 2,500 katals / kg.

[0098] material: Jenway 6305 spectrophotometer (instrument reference) Sigma-Aldrich™ Semi-Micro UV Cuvettes (Z628026) Eppendorf pipette (see Equipment) Peqlab TS-100 Thermoshaker (see Equipment) Thermo Fisher Scientific NanoDrop lite (see Equipment) Sigma-Aldrich (Eppendorf) 3810X Microtube (Z606340) Sigma-Aldrich (GE) BSA (05470) Sigma-AldrichHyaluronic acid (53747) Sigma-AldrichBovine hyaluronidase Type IS (H3506) Carl-Roth Acetate (3738.2) Carl Roth HCl (P074.4) Carl Roth NaOH (9356.1) Carl Roth Sodium Acetate (3856.1) Carl Roth Sodium Phosphate Monobasic (2370.3) Sartorius Arium Mini Plus (see device)

[0099] 1.4.3 Endotoxin quantification For endotoxin quantification, the test kit ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (L00350) by the supplier GenScript was used according to the manufacturer's protocol. The kit contained endotoxin standards, endotoxin-free water, Limulus Amebocyte Lysate, chromogenic substrate, color stabilizer, endotoxin-free pipette tips and tubes, and a tube rack. All packaging was washed with ultrapure water and components were sterilized under laminar flow. Powder / lyophilized components were reconstituted and stored according to the manufacturer's protocol. Four endotoxin measurement standards were prepared by diluting the endotoxin standard of the supplied kit (0.1EU / ml, 0.25EU / ml, 0.5EU / ml, 1.0EU / ml). The standards were used to generate a reference curve that allowed the absorbance of each sample to be converted to a concentration in EU / ml. During every batch sample measurement, the respective standards were analyzed in parallel. For batch analysis, a single batch analytical aliquot was thawed on ice, the tubes were sanitized, washed with ultrapure water and transferred to laminar flow. 100 μl of undiluted batch sample, 100 μl of endotoxin-free water as blank, and four standards with endotoxin concentrations between 0.1 EU / ml and 1.0 EU / ml were used in one analytical run. All sample handling was performed under sterile laminar flow conditions. The following steps were performed: 1. Incubate thoroughly mixed standards, blanks and samples with LAL for 12 minutes at 37°C. 2. Add substrate solution for color reaction and incubate at 37°C for 6 minutes. 3. Add the three color stabilizer solutions stepwise to each measurement vial while gently mixing. 4. The final reaction solution is transferred to a cuvette for absorbance measurement at 545 nm in a spectrophotometer. The background from the blank was subtracted from the sample measurements of all batches and a standard curve was plotted. Endotoxin concentrations of the samples in EU / ml were extrapolated from the standard curve. Endotoxin levels were further stated as EU / mg of product, considering a final batch product / aliquot concentration of 0.1 mg / ml.

[0100] 1.4.4 Sterility testing Vegetable LB agar (soy peptone 10 g / l, NaCl 10 g / l, yeast extract 5 g / l, agar 15 g / l) was prepared with ultrapure water and autoclaved. The hot bottles were placed in an ethanol sterilized laminar flow together with sterile Petri dishes. No antibiotics were added to the growth medium and the plates were poured. For sterility testing, single batch analytical aliquots were removed and thawed on ice. After the plates were solidified and cooled, single batch analytical aliquots were transferred onto single plates with a pipette with a sterile pyrogen-free pipette tip and spread by a sterile Lazy-L-Spreader. A plate containing sterile filtered Tris-HCl NaCl served as a control to test for environmental contamination. A plate containing untransformed E. coli BL21(DE3) served as a positive control. The plates were closed but not sealed to allow air exchange. To ensure environmental sterility, product sterility test plates were kept in laminar flow for 4 days and checked for any growth (time frame may be adapted depending on the rate of environmental contamination during laminar flow processing). Product sterility is confirmed by the absence of visible growth.

[0101] Figure 3 shows images of LB agar plates over a four day period, including an E. coli positive control (Figure 3a), an LB plate negative control (Figure 3b), and a d016 sample of the present invention (Figure 3c). The modified bacterial hyaluronidase d016 of the present invention did not show any growth in antibiotic-free vegetative LB agar plates over a four day period and should therefore be considered to be sterile.

[0102] Thus, the modified bacterial hyaluronidase d016 of the present invention can be used in pharmaceutical compositions, particularly pharmaceutical compositions requiring sterile qualities, such as intravenous applications.

[0103] material: Carl Roth Vegetable Peptone (2832.2) Carl Roth NaCl (3957.3) Carl Roth yeast extract (2363.3) Carl Roth agar (6494.1) Sigma Aldrich (sterile) Petri dishes (P5981) Sigma Aldrich (sterile) Lazy-L-Spreaders (Z376779) Carl Roth (Sorenson Bioscience) Pyrogen-Free 1000μl Filter Tip (9773.1) Eppendorf pipette (see Equipment)

[0104] 1.4.5 Stability and solubility, freeze-thaw testing To test the stability and solubility of the inventive samples, specific activity measurements of multiple concentrations of purified protein were performed over time. Multiple sample dilutions were prepared in sterile filtered Tris HCl NaCl at 0.2 mg / ml (200% final stock solution of product), 0.1 mg / ml (target final stock solution of product) and 0.01 mg / ml (low concentration dose for the target application). The inventive protein concentration was measured using 280 nm absorbance. Each dilution was stored at -15°C, 2-8°C and 25°C, which represent appropriate storage and handling conditions. Aliquots for specific activity measurements were removed at the start of the stability study, after 1 day, 2 days, 3 days, 1 week, 2 weeks and 4 weeks. All aliquots were stored in Tris HCl NaCl with a NaCl concentration of 154 mM, which is equivalent to the ionic strength of 0.9% medicinal saline. The aliquots were also stored on ice until measurement. Measurements were performed according to the activity measurement protocol.

[0105] Additionally, freeze-thaw studies were performed to benchmark how the product solution resisted ice crystal formation during the freeze-thaw process. Samples were frozen and thawed at -80°C five times while aliquots were removed after each cycle to measure activity. General stability and solubility results demonstrated that specific activity did not decrease overall for up to 7 days when sample concentrations were greater than 0.1 mg / ml at -15°C, 2-8°C, and 25°C. Freeze-thaw stability for -80°C freeze-thaw cycles does not decrease specific activity if samples are frozen less than twice. Sample storage concentrations showed a loss of 5-15% protein during one freeze-thaw cycle and during one day of storage at -15°C and 2-8°C. This is likely due to adsorption. Samples with concentrations greater than 0.2 mg / ml at 25°C do not appear to show concentration loss over 7 days.

[0106] The results show that the final product should be stored in protein low binding tubes, preferably at a concentration above 0.2 mg / ml, and not frozen more than twice to ensure absolute stability. Measurements were performed in small microtubes (1.5 ml) with small filling volumes of 150-1500 μl. This results in a much smaller volume-to-surface ratio compared to storage of the product in 50 ml tubes. Therefore, the results above are very likely to be more dependent on adsorption effects than the commercial product.

[0107] material: Sigma-Aldrich Tris (TRIS-RO) Carl Roth HCl (P074.4) Carl Roth NaCl (3957.3) Thermo Fisher Scientific NanoDrop lite (see Equipment) Sigma-Aldrich (Eppendorf) 3810X Microtube (Z606340) Eppendorf pipette (see Equipment)

[0108] 2: Comparison of specific activity between prior art hyaluronidase peptides and the modified bacterial hyaluronidase of the present invention (syn:d016) 2.1 Comparison of the specific activity of bacterial hyaluronidase from Streptomyces koganeiensis (syn:Messina hyaluronidase) and the modified bacterial hyaluronidase of the present invention "rHyal_Sk in the soluble part of the periplasm was thus produced at a final concentration of approximately 2 g / L of culture medium with a very high functional activity (more than 40,000 units / mg), 670-750 times higher than the autologous Hyal produced by fermentation" (see Messina et al., "Identification and characterization of a bacterial hyaluronidase and its production in recombinant form", Federation of European Biochemical Societies (FEBS) Letters, Volume 590, Issue 14, July 2016, pp. 2180-2189).

[0109] Therefore, the specific activity per mg of bacterial Messina hyaluronidase from Streptomyces koganeiensis is greater than 40,000 U / mg, i.e., between 40,000 U / mg and 50,000 U / mg.

[0110] The specific activity of the modified bacterial hyaluronidase d016 of the present invention shown in Example 1.4.2 is 1,500,000 U / mg.

[0111] Thus, the specific activity of the modified bacterial hyaluronidase d016 of the present invention per mg of protein is approximately 30-37.5 times that of the comparative Messina hyaluronidase.

[0112] 2.2 Comparison of Human PH20 Hyaluronidase with Modified Bacterial Hyaluronidase d016 of the Present Invention "The activity of Human PH20, His-Tag (Cat. No. PH0-H5225) is measured by its ability to hydrolyze HA in a turbidimetric assay (45 min assay). Its specific activity is >40,000 U / mg. (Unit definition: One unit of hyaluronidase activity causes a change in A600 of 0.330 per minute in a 2.0 mL reaction mixture at 37°C, pH 5.35)" - ACRObiosystems, https: / / www.acrobiosystems.com / P563-Human-PH20--SPAM1-Protein-His-Tag.html.

[0113] Thus, the specific activity per mg of human PH20 hyaluronidase is greater than 40,000 U / mg, i.e., between 40,000 U / mg and 50,000 U / mg.

[0114] The specific activity of the modified bacterial hyaluronidase d016 of the present invention shown in Example 1.4.2 is 1,500,000 U / mg.

[0115] Thus, the specific activity of the modified bacterial hyaluronidase d016 of the present invention per mg of protein is approximately 30-37.5 times that of the comparative human PH20 hyaluronidase.

[0116] 2.3 Comparison of bovine hyaluronidase with the modified bacterial hyaluronidase d016 of the present invention "Hyaluronidases degrade hyaluronan and have been found to be inappropriately regulated during cancer progression. These enzymes randomly cleave the β-N-acetylhexosamine-[1→4] glycosidic bonds of hyaluronic acid, chondroitin, and chondroitin sulfate. Unit definition: 1 unit causes a % change in transmittance at 600 nm of 0.330 per minute at pH 5.35 and 37°C in a 2.0 mL reaction mixture (45 minute assay)." -See Sigma-Aldrich, supplier of bovine testicular hyaluronidase. Bovine testicular hyaluronidase, IS type, freeze-dried powder, 400-1,000U / mg solid, Bovine testicular hyaluronidase suitable for mouse embryonic cell culture, type IV-S, powder, 750-3,000U / mg solid, Bovine testicular hyaluronidase, type IV-S, lyophilized powder (essentially salt-free), 750-3,000U / mg, Bovine testicular hyaluronidase, type VIII, lyophilized powder, 300-1,000U mg, Bovine testicular hyaluronidase, type VI-S, lyophilized powder, 3000~15,000 U / mg.

[0117] Therefore, the specific activity of bovine hyaluronidase ranges widely from 300 to 15,000 U / mg.

[0118] The specific activity of the modified bacterial hyaluronidase d016 of the present invention shown in Example 1.4.2 is 1,500,000 U / mg.

[0119] Therefore, the specific activity of the modified bacterial hyaluronidase d016 of the present invention per mg of protein is approximately 100 to 5,000 times that of the comparative bovine hyaluronidase.

Claims

1. A modified bacterial hyaluronidase polypeptide comprising, or consisting of, SEQ ID NO:1, wherein the hyaluronidase polypeptide comprises a C-terminal HIS tag of SEQ ID NO:7 and an N-terminal Strep tag of SEQ ID NO:

5.

2. The modified bacterial hyaluronidase polypeptide described in claim 1 for use in the treatment or prevention of a disease or disorder selected from the group consisting of homozygous familial hypercholesterolemia (xanthomatosis), heterozygous familial hypercholesterolemia and diabetic foot.

3. A nucleic acid encoding the modified bacterial hyaluronidase polypeptide of claim 1.

4. A recombinant expression vector comprising an expression vector and the nucleic acid of claim 3.

5. A host cell transformed with the recombinant expression vector of claim 4.

6. 3. A method for producing a purified bacterial hyaluronidase polypeptide according to claim 1 or 2, comprising the steps of: a. culturing the transformed host cell of claim 5 in a suitable growth medium under suitable growth conditions to express the bacterial hyaluronidase polypeptide of claim 1 or 2; b. harvesting the cultured transformed host cells of step a); c. lysing the harvested host cells of step b) and separating the resulting host cell fragments from the resulting host cell contents that contain the bacterial hyaluronidase polypeptide; and d. purifying the resulting host cell content of step c) using HIS affinity chromatography and STREP affinity chromatography to obtain a purified form of the bacterial hyaluronidase polypeptide of claim 1 or 2; A method comprising or consisting of the steps of:

7. The method of claim 6, wherein the host cell is E. coli.

8. A modified bacterial hyaluronidase polypeptide comprising or consisting of SEQ ID NO:1, obtained according to the production method of claim 6 or 7.

9. 10. A pharmaceutical composition comprising a therapeutically effective amount of a modified bacterial hyaluronidase polypeptide of any one of claims 1, 2 and 8, and one or more pharma- ceutically acceptable excipients.

10. 10. The pharmaceutical composition of claim 9, wherein a unit dose of the composition comprises the modified bacterial hyaluronidase polypeptide in an amount of 200 U / kg / day to 30,000 U / kg / day.

11. 11. The pharmaceutical composition according to claim 9 or 10, wherein the composition is selected from a solid, semi-solid or liquid application form.

12. The pharmaceutical composition according to any one of claims 9 to 11, wherein said composition is suitable for oral, nasal, transdermal, rectal, intravenous or intramuscular application.

13. A pharmaceutical composition described in any one of claims 9 to 12 for use in the treatment or prevention of a disease or disorder selected from the group consisting of homozygous familial hypercholesterolemia (xanthomatosis), heterozygous familial hypercholesterolemia, and diabetic foot.

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