阅读说明：本技术 一种重组人透明质酸酶的液体制剂及其应用 (Liquid preparation of recombinant human hyaluronidase and application thereof ) 是由 王征 楼俊文 房鑫 叶亚文 胡颖 唐瑶 于 2020-06-11 设计创作，主要内容包括：本发明公开了一种重组人透明质酸酶液体制剂及其应用,包含：重组人透明质酸酶、缓冲剂、稳定剂和非离子型表面活性剂,稳定剂选自海藻糖、蔗糖、甘露醇、氯化钠、甲硫氨酸中的一种或多种,可维持重组人透明质酸酶在2～8℃条件下的稳定性。该制剂既可单独施用,又可和其他制剂同时或者先后施用,又或者和其他制剂混合后施用,临床使用较冻干制剂更加方便。(The invention discloses a recombinant human hyaluronidase liquid preparation and application thereof, comprising: the recombinant human hyaluronidase inhibitor comprises recombinant human hyaluronidase, a buffering agent, a stabilizing agent and a non-ionic surfactant, wherein the stabilizing agent is selected from one or more of trehalose, sucrose, mannitol, sodium chloride and methionine, and can maintain the stability of the recombinant human hyaluronidase at the temperature of 2-8 ℃. The preparation can be used independently, simultaneously or sequentially with other preparations, or mixed with other preparations for administration, and is more convenient for clinical use than lyophilized preparation.)

1. A liquid formulation of recombinant human hyaluronidase enzyme comprising: recombinant human hyaluronidase, a buffer, a stabilizer, and a non-ionic surfactant, wherein:

the enzyme activity of the recombinant human hyaluronidase is 45 units/ml-4500000 units/ml;

the concentration of the buffer is 1-100 mM;

the concentration of the stabilizer is 1-200 mM, and the stabilizer is selected from one or more of trehalose, sucrose, mannitol, sodium chloride and methionine;

the concentration of the nonionic surfactant is 0.01-0.1% (w/v);

the pH value of the liquid preparation is 5.5-8.0.

2. The liquid formulation of claim 1, wherein the stabilizing agent is selected from one or more of trehalose, sodium chloride and methionine, preferably methionine and/or sodium chloride, such as methionine, sodium chloride, or a combination of both methionine and sodium chloride; preferably:

the trehalose concentration is 10-250 mM, preferably 20-200 mM, such as 25mM, 53mM or 200 mM;

and/or the concentration of the sodium chloride is 25-200 mM, preferably 30-200 mM, such as 30mM, 130mM, 145mM or 180 mM;

and/or the concentration of methionine is 5-50 mM, preferably 5mM, 10mM or 50 mM;

when the stabilizing agent is methionine and sodium chloride, the concentration of the methionine is 5-15 mM, and the concentration of the sodium chloride is 50-180 mM.

3. The liquid preparation according to claim 1, wherein the recombinant human hyaluronidase has an enzymatic activity of 45 units/ml to 3000000 units/ml, preferably 45 units/ml to 1500000 units/ml, more preferably 45 units/ml to 300000 units/ml.

4. The liquid preparation according to claim 1, wherein the buffer is one or more of histidine buffer, acetate buffer, phosphate buffer, citrate buffer, Tris buffer; and/or, the non-ionic surfactant is selected from one or more of polysorbate 20, polysorbate 80 and poloxamer 188;

wherein:

the concentration of the acetic acid buffer solution is preferably 5-50 mM, such as 5mM, 10mM or 50 mM;

the concentration of the phosphate buffer solution is preferably 5-50 mM, such as 5mM, 10mM or 50 mM;

the concentration of the citric acid buffer solution is preferably 5-50 mM, such as 5mM, 10mM or 50 mM;

the concentration of the Tris buffer solution is preferably 5-50 mM, such as 5mM, 10mM or 50 mM;

the concentration of the nonionic surfactant is preferably 0.02% (w/v).

5. The liquid formulation of claim 1, wherein the recombinant human hyaluronidase liquid formulation is one of the following compositions:

(1) 45-4500000 units/ml of recombinant human hyaluronidase, 10mM of phosphate buffer, 145mM of sodium chloride, 10mM of methionine, 0.02% of polysorbate 20, and the pH value of the mixture is 6.5-7.5;

(2) 45-4500000 units/ml of recombinant human hyaluronidase, 10mM of phosphate buffer, 130mM of sodium chloride, 53mM of trehalose, 10mM of methionine, 0.02% of polysorbate 20, and pH of 6.5-7.5;

(3) 45-4500000 units/ml of recombinant human hyaluronidase, 10mM of phosphate buffer, 130mM of sodium chloride, 53mM of sucrose, 10mM of methionine, 0.02% of polysorbate 20, and pH of 6.5-7.5;

(4) 45-4500000 units/ml of recombinant human hyaluronidase, 50mM histidine buffer, 130mM sodium chloride, 53mM trehalose, 10mM methionine, 0.02% polysorbate 20, and pH 5.5-6.5;

(5) 45-4500000 units/ml of recombinant human hyaluronidase, 50mM acetic acid buffer, 130mM sodium chloride, 53mM trehalose, 10mM methionine, 0.02% polysorbate 20, and pH 5.0-6.0;

(6) 45-4500000 units/ml of recombinant human hyaluronidase, 50mM of citric acid buffer, 130mM of sodium chloride, 53mM of trehalose, 10mM of methionine, 0.02% of polysorbate 20, and pH of 6.0-7.0;

(7) 45-4500000 units/ml of recombinant human hyaluronidase, 50mM Tris buffer, 130mM sodium chloride, 53mM trehalose, 10mM methionine, 0.02% polysorbate 20, and pH 7.0-8.0;

(8) 45-4500000 units/ml of recombinant human hyaluronidase, 10mM of phosphate buffer, 30mM of sodium chloride, 200mM of trehalose, 10mM of methionine, 0.02% of polysorbate 20, and pH 6.5-7.5;

(9) 45-4500000 units/ml of recombinant human hyaluronidase, 10mM of phosphate buffer, 180mM of sodium chloride, 25mM of trehalose, 10mM of methionine, 0.02% of polysorbate 20, and pH 6.5-7.5;

(10) 45-4500000 units/ml of recombinant human hyaluronidase, 10mM of phosphate buffer, 30mM of sodium chloride, 200mM of sucrose, 10mM of methionine, 0.02% of polysorbate 20, and pH 6.5-7.5;

(11) 45-4500000 units/ml of recombinant human hyaluronidase, 10mM of phosphate buffer, 180mM of sodium chloride, 25mM of sucrose, 10mM of methionine, 0.02% of polysorbate 20, and pH 6.5-7.5;

(12) 45-4500000 units/ml of recombinant human hyaluronidase, 10mM of phosphate buffer, 130mM of sodium chloride, 53mM of trehalose, 5mM of methionine, 0.02% of polysorbate 20, and pH of 6.5-7.5;

(13) 45-4500000 units/ml of recombinant human hyaluronidase, 10mM of phosphate buffer, 130mM of sodium chloride, 53mM of trehalose, 50mM of methionine, 0.02% of polysorbate 20, and pH of 6.5-7.5;

(14) 45-4500000 units/ml of recombinant human hyaluronidase, 10mM of phosphate buffer, 130mM of sodium chloride, 53mM of trehalose, 10mM of methionine, 0.02% of polysorbate 80, and pH of 6.5-7.5;

(15) 45-4500000 units/ml of recombinant human hyaluronidase, 10mM of phosphate buffer, 130mM of sodium chloride, 53mM of trehalose, 10mM of methionine, 0.02% of poloxamer 188 and pH 6.5-7.5.

6. A kit of parts comprising the liquid formulation of any one of claims 1 to 5, further comprising a therapeutic agent;

the therapeutic agent is preferably a recombinant protein, a fusion protein, human serum albumin, an immunoglobulin, a targeted antibody drug, a polypeptide, a nanoparticle, a virus, a cell, or a chemical;

wherein the polypeptide is preferably insulin, a glucagon-like peptide-1 (GLP-1) analog, leuprolide, vasopressin or bacitracin;

or the therapeutic agent is preferably a drug having an anti-tumor effect;

the drug with the anti-tumor effect is preferably one or more of oncolytic virus, immune cell, CAR-T cell, TCR-T cell, anti-tumor chemical drug, tumor-targeted antibody drug, ADC antibody drug, immune checkpoint inhibitor, nanoparticle drug and corticosteroid;

the tumor-targeting antibody drug is preferably one or two of trastuzumab, rituximab, pertuzumab, daratuximab and Isatuximab; such as rituximab, trastuzumab, and pertuzumab;

when the tumor-targeting antibody drug is pertuzumab or trastuzumab, the dose of the pertuzumab is 600mg or 1200mg, and the dose of the trastuzumab is 600 mg;

when the tumor-targeted antibody drug is rituximab, the dose of the rituximab is 1400mg or 1600 mg;

when the tumor-targeting antibody drug is trastuzumab, the dose of the trastuzumab is 600 mg.

7. A method for treating a disease, characterized by administering to a subject a liquid formulation of recombinant human hyaluronidase according to any one of claims 1-5 or a kit of parts according to claim 6;

the administration is preferably separate or mixed.

8. The method of claim 7, wherein said separate administrations comprise the steps of:

(a) administering intradermally or subcutaneously to a subject the liquid formulation of recombinant human hyaluronidase in the kit; and

(b) subsequently administering to the subject the therapeutic agent in the kit;

wherein steps (a) and (b) are performed separately, simultaneously or alternately; preferably:

when steps (a) and (b) are carried out separately, the time interval between steps (a) and (b) is 0-24 hours;

preferably no time interval, at most 1 minute, at most 2 minutes, at most 3 minutes, at most 4 minutes, at most 5 minutes, at most 6 minutes, at most 7 minutes, at most 8 minutes, at most 9 minutes, at most 10 minutes, at most 15 minutes, at most 20 minutes, at most 25 minutes, at most 30 minutes, at most 1h, at most 2h, at most 3h, at most 6h, at most 12h or at most 24 h.

9. The method according to claim 8, wherein the time interval between steps (a) and (b) is less than 10 minutes, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 minutes, when administered subcutaneously in succession.

10. The method of claim 8 or 9, wherein said separate administrations are effected simultaneously or sequentially by means of a triplet;

the respective administration can be controlled by an infusion pump or gravity;

wherein the liquid preparation of the recombinant human hyaluronidase is administered at a rate of 0.1-2 ml/min;

the therapeutic agent is infused at a rate of 5 ml/hour, 10 ml/hour, 30 ml/hour, 60 ml/hour, 120 ml/hour, 240 ml/hour, or 300 ml/hour.

11. The method of claim 7, wherein said co-administering comprises: administering intradermally or subcutaneously to the subject after mixing the liquid formulation of recombinant human hyaluronidase in the kit with a therapeutic agent;

the therapeutic agent for mixing is preferably in liquid or dry powder form;

wherein, the mixed administration can be controlled by an infusion pump or gravity;

the liquid preparation of the recombinant human hyaluronidase and the therapeutic agent are mixed and then infused at a rate of 5-300 ml/hour, such as 5 ml/hour, 10 ml/hour, 30 ml/hour, 60 ml/hour, 120 ml/hour, 240 ml/hour or 300 ml/hour.

12. The method according to any one of claims 7 to 11, wherein the subject is a subject who has undergone surgery or trauma followed by local edema or hematoma, receives local anesthesia, receives infusion or receives injection of the contrast agent barium sulfate;

preferably, the subject is suffering from local ocular depot, exudate or blood, vitreous opacity or chemical conjunctival burn with inflammation, traumatic orbital bleeding or traumatic retinal edema.

13. The method of any one of claims 7-11, wherein the liquid formulation of recombinant human hyaluronidase is administered directly, and/or after dilution;

wherein the diluting preparation is preferably physiological saline;

the dilution ratio is preferably 1: 10.

14. A subcutaneous delivery device comprising a liquid formulation of the recombinant human hyaluronidase of any one of claims 1-5;

the subcutaneous delivery device comprises: a container, a seal and an injection needle;

wherein:

the container is preferably a vial, a syringe or a test tube;

the sealing element is preferably a sealing plug or a sealing ring;

the injection needle is preferably a water needle or a single needle-micro needle set.

15. The subcutaneous delivery device as in claim 14, wherein said container is made of glass or plastic;

and/or the sealing element is made of rubber, plastic or high polymer material;

the material of the injection needle is metal, silicon dioxide, glass, nickel, titanium or biodegradable polymer.

16. The subcutaneous delivery device as claimed in claim 14 or 15, wherein said hydro-needle comprises: a vial water needle, an ampoule water needle or a pre-filling injection system;

wherein, the ampoule bottle is preferably a glass ampoule or a plastic ampoule;

the prefilled injection system is preferably a prefilled syringe, such as a prefilled needle, a prefilled syringe, or a prefilled cartridge.

Technical Field

The invention relates to the field of biomedicine, in particular to a liquid preparation of recombinant human hyaluronidase and application thereof.

Background

Hyaluronic Acid (HA), also known as uronic acid, hyaluronic acid or hyaluronic acid, is a linear high molecular glycosaminoglycan composed of two repetitive disaccharide units, D-glucuronic acid and N-acetylglucosamine, widely found in connective tissues, mucus tissues, lens of eyeball and skin of vertebrates, and particularly abundant in tissues such as embryo, cartilage, synovial fluid, vitreous body, umbilical cord, comb, etc. Hyaluronic acid is an acidic mucopolysaccharide widely distributed in human tissue matrixes, and has multiple functions in vivo, such as forming multiple matrixes, limiting the diffusion of water and other extracellular substances, regulating osmotic pressure, regulating the transport of macromolecular substances, forming a physical barrier around cells, and the like. As hyaluronic acid fills between extracellular matrix collagen fibrous scaffolds in vivo by forming a reticulated barrier, subcutaneous diffusion of drug macromolecular species is restricted, the rate of drug arrival at the blood vessel is reduced, and the volume of liquid injected subcutaneously is limited.

Hyaluronidase (also called hyaluronidase) is an endoprotease that specifically hydrolyzes hyaluronic acid and thereby increases the ability of fluid penetration in tissues, and is widely distributed in vivo in tissues and body fluids such as serum, brain, and placenta. Hydrolysis of hyaluronic acid by breaking the gluconic acid bond between glucosamine C1 and glucuronic acid C4 temporarily reduces the viscosity of the intercellular substance, promotes the diffusion of subcutaneous infusion, locally stored exudate or blood, and facilitates absorption.

Generally, commercially available hyaluronidases are obtained by salting out mammalian testis and then lyophilizing, or by removing pyrogens after salting out and then lyophilizing. For example, the following materials can be used as raw materials for the purification process: mammalian testicular material salted once and then lyophilized, testicular material salted twice and then lyophilized, or testicular material salted twice, depyrogenated, and then lyophilized. The existing hyaluronidase is extracted by twice salting out from sheep testis, freeze-drying, dialyzing, depyrogenating, and then freeze-drying, and the extracted material incorporates a large amount of proteins other than hyaluronidase.

Commercially available lyophilized hyaluronidase in a crude state contains a large amount of impurities, has low purity, and is poor in stability when prepared into a liquid formulation. In addition, the freeze-dried preparation needs to be prepared into a liquid form before use, so that the clinical use is inconvenient, and the medical care cost is increased.

CN104768535B discloses a chelating agent and MgCl₂The hyaluronidase (animal origin) liquid preparation of (1), wherein the hyaluronidase activity has poor stability at a temperature of 2-8 ℃ relative to the initial enzyme activity of 100 hyaluronidase, and only 90% of the hyaluronidase activity is maintained after 29 weeks. Another prior art is Hylenex medicine of Halozyme company, which is a liquid preparation, wherein human serum albumin with the content of 1mg/ml is used in the liquid preparation, and the human serum albumin has high cost, is derived from blood plasma and has high risk of infection by pathogens.

Therefore, a liquid formulation comprising hyaluronidase with superior stability is urgently needed for convenience of clinical use and reduction of risk.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the prior art and provide a recombinant human hyaluronidase liquid preparation and application thereof, wherein the preparation is convenient to use clinically, has wide application, simple formula and good product stability, and the stability is kept above 95% after the preparation is placed at the temperature of 2-8 ℃ for 12 months.

The invention is realized by the following technical scheme:

a first aspect of the present invention relates to a recombinant human hyaluronidase liquid formulation comprising: recombinant human hyaluronidase, a buffer, a stabilizer, and a non-ionic surfactant, wherein:

the enzyme activity of the recombinant human hyaluronidase is 50-4500000 units/ml;

the concentration of the buffer is 1-100 mM;

the concentration of the stabilizer is 1-200 mM, and the stabilizer is selected from one or more of trehalose, sucrose, mannitol, sodium chloride and methionine;

the concentration of the nonionic surfactant is 0.01-0.1% (w/v);

the pH value of the liquid preparation is 5.5-8.0.

The enzyme activity of the recombinant human hyaluronidase is preferably 45 units/ml-3000000 units/ml; more preferably 45 units/ml to 1500000 units/ml; further preferably 45 units/ml to 300000 units/ml, for example 45 units/ml, 100 units/ml, 150 units/ml, 500 units/ml, 1000 units/ml, 5000 units/ml, 50000 units/ml, 300000 units/ml, 1500000 units/ml, 3000000 units/ml or 4500000 units/ml.

The concentration of the recombinant human hyaluronidase is preferably 0.15-0.75. mu.g/ml, 0.75-2.5. mu.g/ml, 2.5-5. mu.g/ml, 5.5-25. mu.g/ml, 25. mu.g/ml-0.3 mg/ml, 0.3-1.5 mg/ml, 1.5-7.5 mg/ml, 7.5-15 mg/ml or 15-22.5 mg/ml, such as 0.15. mu.g/ml, 0.33. mu.g/ml, 0.5. mu.g/ml, 1.7. mu.g/ml, 3.3. mu.g/ml, 17. mu.g/ml, 0.2mg/ml, 1mg/ml, 5mg/ml, 10mg/ml or 15 mg/ml.

The recombinant human hyaluronidase has enzyme activity of 300000 units/ml-1500000 units/ml, 1500000 units/ml-3000000 units/ml or 300000 units/ml-450000 units/ml.

Preferably, the buffer provides a pH of 5.5 to 7.5, such as 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0.

The buffer may be conventional in the art, and is preferably one or more of histidine buffer, acetate buffer, phosphate buffer, citrate buffer, Tris buffer, such as histidine buffer, acetate buffer, phosphate buffer, citrate buffer, or Tris buffer.

The pH value of the histidine buffer solution is 5.5-7.5, the pH value of the acetic acid buffer solution is 5.0-6.0, the pH value of the phosphate buffer solution is 6.0-8.0, the pH value of the citric acid buffer solution is 5.0-7.0, and the pH value of the Tris buffer solution is 7.0-8.0.

Preferably, the pH of the histidine buffer is 6.0.

Preferably, the pH of the acetic acid buffer is 5.0.

Preferably, the pH of the phosphate buffer is 7.0.

Preferably, the pH of the citric acid buffer is 6.5.

Preferably, the Tris buffer has a pH of 8.0.

Preferably, the histidine buffer concentration is 5-50 mM, such as 5mM, 10mM or 50 mM.

Preferably, the concentration of the acetic acid buffer is 5-50 mM, such as 5mM, 10mM or 50 mM.

Preferably, the phosphate buffer is at a concentration of 5 to 50mM, such as 5mM, 10mM or 50 mM.

Preferably, the concentration of the citric acid buffer is 5-50 mM, such as 5mM, 10mM or 50 mM.

Preferably, the Tris buffer concentration is 5-50 mM, such as 5mM, 10mM or 50 mM.

Preferably, the stabilizer is selected from one or more of trehalose, sodium chloride and methionine; more preferably, the stabilizer is methionine and/or sodium chloride.

Preferably, the trehalose concentration is 25-200 mM, such as 25mM, 53mM or 200 mM.

Preferably, the sodium chloride concentration is 30-200 mM, such as 30mM, 130mM, 145mM or 180 mM.

Preferably, the methionine concentration is 5-50 mM, such as 5mM, 10mM or 50 mM.

When the stabilizing agent is methionine and sodium chloride, the concentration of the methionine is 5-15 mM, and the concentration of the sodium chloride is 50-180 mM.

The nonionic surfactant is one selected from polysorbate 20, polysorbate 80 and poloxamer 188.

Preferably, the concentration of the nonionic surfactant is 0.02% (w/v).

In a preferred embodiment of the present invention, the liquid preparation of recombinant human hyaluronidase comprises one of the following compositions:

(1) 45-4500000 units/ml (about 0.15-15 mg/ml) of recombinant human hyaluronidase, 10mM phosphate buffer, 145mM sodium chloride, 10mM methionine, 0.02% polysorbate 20, pH 6.5-7.5.

(2) 45-4500000 units/ml (about 0.15-15 mg/ml) of recombinant human hyaluronidase, 10mM phosphate buffer, 130mM sodium chloride, 53mM trehalose, 10mM methionine, 0.02% polysorbate 20, pH 6.5-7.5.

(3) 45-4500000 units/ml (about 0.15-15 mg/ml) of recombinant human hyaluronidase, 10mM phosphate buffer, 130mM sodium chloride, 53mM sucrose, 10mM methionine, 0.02% polysorbate 20, pH 6.5-7.5.

(4) 45-4500000 units/ml (about 0.15-15 mg/ml) of recombinant human hyaluronidase, 50mM histidine buffer, 130mM sodium chloride, 53mM trehalose, 10mM methionine, 0.02% polysorbate 20, pH 5.5-6.5.

(5) 45-4500000 units/ml (about 0.15-15 mg/ml) of recombinant human hyaluronidase, 50mM acetate buffer, 130mM sodium chloride, 53mM trehalose, 10mM methionine, 0.02% polysorbate 20, pH 5.0-6.0.

(6) 45-4500000 units/ml (about 0.15-15 mg/ml) recombinant human hyaluronidase, 50mM citric acid buffer, 130mM sodium chloride, 53mM trehalose, 10mM methionine, 0.02% polysorbate 20, pH 6.0-7.0.

(7) 45-4500000 units/ml (about 0.15-15 mg/ml) recombinant human hyaluronidase, 50mM Tris buffer, 130mM sodium chloride, 53mM trehalose, 10mM methionine, 0.02% polysorbate 20, pH 7.0-8.0.

(8) 45-4500000 units/ml (about 0.15-15 mg/ml) of recombinant human hyaluronidase, 10mM phosphate buffer, 30mM sodium chloride, 200mM trehalose, 10mM methionine, 0.02% polysorbate 20, pH 6.5-7.5.

(9) 45-4500000 units/ml (about 0.15-15 mg/ml) recombinant human hyaluronidase, 10mM phosphate buffer, 180mM sodium chloride, 25mM trehalose, 10mM methionine, 0.02% polysorbate 20, pH 6.5-7.5.

(10) 45-4500000 units/ml (about 0.15-15 mg/ml) of recombinant human hyaluronidase, 10mM phosphate buffer, 30mM sodium chloride, 200mM sucrose, 10mM methionine, 0.02% polysorbate 20, pH 6.5-7.5.

(11) 45-4500000 units/ml (about 0.15-15 mg/ml) recombinant human hyaluronidase, 10mM phosphate buffer, 180mM sodium chloride, 25mM sucrose, 10mM methionine, 0.02% polysorbate 20, pH 6.5-7.5.

(12) 45-4500000 units/ml (about 0.15-15 mg/ml) of recombinant human hyaluronidase, 10mM phosphate buffer, 130mM sodium chloride, 53mM trehalose, 5mM methionine, 0.02% polysorbate 20, pH 6.5-7.5.

(13) 45-4500000 units/ml (about 0.15-15 mg/ml) of recombinant human hyaluronidase, 10mM phosphate buffer, 130mM sodium chloride, 53mM trehalose, 50mM methionine, 0.02% polysorbate 20, pH 6.5-7.5.

(14) 45-4500000 units/ml (about 0.15-15 mg/ml) of recombinant human hyaluronidase, 10mM phosphate buffer, 130mM sodium chloride, 53mM trehalose, 10mM methionine, 0.02% polysorbate 80, pH 6.5-7.5.

(15) 45-4500000 units/ml (about 0.15-15 mg/ml) of recombinant human hyaluronidase, 10mM phosphate buffer, 130mM sodium chloride, 53mM trehalose, 10mM methionine, 0.02% poloxamer 188, pH 6.5-7.5.

The recombinant human hyaluronidase is human testicular hyaluronidase, preferably, a human testicular hyaluronidase extramembranous domain.

The recombinant human hyaluronidase comprises an amino acid sequence shown as SEQ ID NO.1, such as PH20, and has an amino acid sequence shown as SEQ ID NO. 2.

SEQ ID NO.1

LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEE

SEQ ID NO.2

LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY

A second aspect of the invention provides a kit of parts comprising a liquid formulation of recombinant human hyaluronidase as described above and a therapeutic agent.

The therapeutic agent can be recombinant protein, fusion protein, human serum albumin, immunoglobulin, targeted antibody drug, polypeptide, nanoparticle, virus, cell or chemical drug.

Preferably, the polypeptide can be insulin, a glucagon-like peptide-1 (GLP-1) analog, leuprolide, vasopressin, bacitracin, and the like.

Alternatively, the therapeutic agent is preferably a drug having an antitumor effect.

The drug having an anti-tumor effect may be conventional in the art, such as one or more of an oncolytic virus, an immune cell, a CAR-T cell, a TCR-T cell, an anti-neoplastic drug, a tumor-targeting antibody drug, an ADC antibody drug, an immune checkpoint inhibitor, a nanoparticle drug, and a corticosteroid.

The target of the tumor-targeting antibody drug can be a cell surface protein, such as: AFP, α v integrin (integrin), α 4 β 7 integrin, BCMA, CD2, CD3, CD19, CD20, CD22, CD25, CD30, CD105, CD121, CD123, CD133, CD138, CD174, CD205, CD227, CD326, CD340, CEA, c-Met, Cripto, CA 130, Claudin18.2, EGFR-B, EGFR, EphA 30, EphB 30, FAP, FOLR 30, GD 30, Globo H, GPC 30, GPNMB, HER-1, HER-2, HER-3, MAhA-30, MUSLINX 30, MELR 30, MEWT-30, MGW 30, VEGF-30, MGWT-30, MEWT-30, MGW 30, or GPWT-30.

The target of the antitumor drug can be cytokines, such as: interleukins IL-1 to IL-13, tumor necrosis factors alpha and beta, interferons alpha, beta and gamma, tumor growth factor beta (TGF-beta), Colony Stimulating Factor (CSF) or Granulocyte Monocyte Colony Stimulating Factor (GMCSF). See HumanCytokines: Handbook for Basic & Clinical Research (ed. by Aggrawal et al, Blackwell Scientific, Boston, MA 1991).

The targets of the antitumor drugs can be hormones, enzymes, intracellular and intercellular messengers, such as: adenosine cyclase, guanosine cyclase or phospholipase C.

Preferably, the targeted antibody drug is an antibody drug targeting human epidermal growth factor receptor 2(Her-2), such as trastuzumab, pertuzumab, ZW25, or ZW 49.

Preferably, the targeted antibody drug is an antibody drug targeting CD20, such as rituximab.

Preferably, the targeted antibody drug is an antibody drug targeting CD38, such as daratuzumab or Isatuximab.

Preferably, the effective component of the anti-tumor drug is gemcitabine, paclitaxel, etc.

Preferably, the immune checkpoint inhibitor comprises an immune checkpoint antibody, said immune checkpoint comprising: CTLA-4, PD-1, PD-L1, TIM-3, LAG3, Siglec15, 4-1BB, GITR, OX40, CD40L, CD28, TIGIT, VISTA;

the immune checkpoint antibody can be anti-PD-1 monoclonal antibody, anti-PD-L1 monoclonal antibody, anti-OX-40 monoclonal antibody, anti-CD 47 monoclonal antibody, anti-CTLA-4 monoclonal antibody, anti-TIM-3 antibody, anti-LAG 3 antibody, anti-Siglec 15 antibody, anti-4-1 BB antibody, anti-GITR antibody, anti-OX 40 antibody, anti-CD 40L antibody, anti-CD 28 antibody, anti-TIGIT antibody, anti-VISTA antibody and the like.

Preferably, the antibody is rituximab, trastuzumab, pertuzumab, ZW25, ZW49, daratuximab, and/or Isatuximab.

The targeted antibody drug can also be a non-targeted tumor antibody drug.

The non-targeting tumor antibody drug can be conventional in the art, such as gene therapy virus vector drugs, monoclonal antibodies, bispecific antibodies, multispecific antibodies, chemicals, recombinant proteins, fusion proteins.

In a first embodiment, the kit comprises two single-dose containers, wherein the first container contains a single specific dose of the liquid formulation comprising 45 units/ml to 4500000 units/ml of recombinant human hyaluronidase, and the second container contains a single fixed dose of the therapeutic agent.

The container containing the recombinant human hyaluronidase liquid preparation is selected from a vial and a pre-filling needle; preferably a pre-filled needle.

Preferably, the recombinant human hyaluronidase liquid preparation contains 45 units/ml to 500000 units/ml of recombinant human hyaluronidase.

The volume of the recombinant human hyaluronidase liquid preparation is preferably 0.1-50 ml, such as 0.2ml, 0.5ml, 1ml, 1.5ml, 2ml, 2.50ml, 5.00ml, 10.00ml, 15.00ml, 20ml, 30ml, 40ml or 50 ml.

In some embodiments, the liquid formulation of recombinant human hyaluronidase comprises 45 units to 75000 units of recombinant human hyaluronidase per 1ml, such as 45 units, 100 units, 150 units, 200 units, 500 units, 1000 units, 1500 units, 2000 units, 2500 units, 3000 units, 4000 units, 4500 units, 5000 units, 6000 units, 6500 units, 7000 units, 7500 units, 8000 units, 8500 units, 9000 units, 10000 units, 15000 units, 20000 units, 21000 units, 22000 units, 23000 units, 24000 units, 25000 units, 26000 units, 27000 units, 28000 units, 29000 units, 30000 units, 31000 units, 32000 units, 33000 units, 34000 units, 35000 units, 36000 units, 37000 units, 38000 units, 39000 units, 40000 units, 41000 units, 43000 units, 45000 units, 44000 units, 46000 units, 47000 units, 48000 units, 4900 units, 42000 units, 50000 units, 42000 units, 46000 units, 4700 units, 47000 units, 48000 units, 50000 units, and/ml of the composition, 55000 units, 60000 units, 65000 units, 70000 units, or 75000 units of a recombinant human hyaluronidase.

In a second embodiment, the kit comprises two single dose containers, wherein the first container contains a single fixed dose of the antibody and the second container contains a single specific dose of the liquid formulation comprising 45 units/ml to 4500000 units/ml of recombinant human hyaluronidase.

Wherein, the first container is a tubular bottle, and the second container is a tubular bottle or a pre-filling needle.

Wherein the recombinant human hyaluronidase liquid formulation preferably comprises 45 units/ml to 500000 units/ml of recombinant human hyaluronidase; more preferably, it comprises 45 units/ml to 50000 units/ml of recombinant human hyaluronidase.

The antibody is the targeted antibody drug as described above, and can be: abagovamab, Abciximab, Actoxumab, Adalilimumab, Adecatuzumab, Afelimomab, Afurimomab, Afurimumab, Afurizumab, Anrukinumab, Apolizumab, Arcitmomab, Aselizumab, Atinumab, Atilizumab, Atolizumab (Betacarb), Atorolimumab, Bapineuzumab, Basilizumab, Bavituzumab, Bevituximab, Bevituzumab, Bexizumab, Evituzumab, Avitumomab, Britumomab, Britumom, Glembatumumab, Golmizumab, Gomiliximab, Ibalamazumab, Ibritumumab, Ibrotiuxetan, Ibrucuumamab, Igovatab, Imirosomaab, Iratumumab, Itolizumab, Ikekimab, Iletumumab, Illinumab, Illinumumab, Illinumamab, Illinumab, Illinumamab, Illinuzumab, Lebrinzolumab, Levelumab, Lerdeluzumab, Levellumumab, Ligellifumamab, Ligellifumab, Lipolizumab, Lipurimab, Lintuzumab, Lipurizumab, Lomilizumab, Samilizumab, Illuminazumab, Illuminamab, Lipurimab, secukinumab, Seribantimab, Setoxiximab, Seviruzumab, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitumab, Sonepucimab, Sonepucizumab, Stamuumab, Sulesumab, Suviuzumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talidumab, Tanelumab, Taplimopaptox, Tefibatizumab, Telimeitumab, Telimulizumab, Teplituzumab, Telimumab, Teclizumab, Vesizumab, Vetuzumab, Velutizumab, Vatuzumab, Velutib, Vatuzumab, Velutizumab, Vatuzumab, Velutib, Vatuzumab, Velutizumab, Vatuzumab.

In a third embodiment, the article of manufacture comprises three single-dose containers, wherein the first container contains a single fixed dose of pertuzumab, the second container contains a single fixed dose of trastuzumab, and the third container contains a single specific dose of the liquid formulation comprising 45 units/ml to 4500000 units/ml of recombinant human hyaluronidase.

Wherein, the concentration of the pertuzumab can be 30-150 mg/ml, and is preferably 0-120 mg/ml;

the trastuzumab is a liquid preparation or a freeze-dried preparation; the concentration of the trastuzumab after the liquid preparation and the freeze-dried preparation are re-dissolved can be 20-150 mg/ml, and preferably 20-120 mg/ml;

the recombinant human hyaluronidase liquid preparation preferably comprises 45 units/ml to 500000 units/ml of recombinant human hyaluronidase; more preferably, it comprises 45 units/ml to 50000 units/ml of recombinant human hyaluronidase.

Wherein, the dose of the pertuzumab is preferably 600mg or 1200mg, and the dose of the trastuzumab is preferably 600 mg.

The first container is a tubular bottle, the second container is a tubular bottle, and the third container is a tubular bottle or a pre-filling needle.

In a fourth embodiment, the kit comprises two single-dose containers, wherein the first container contains a single specified dose of the liquid formulation comprising 45 units/ml to 500000 units/ml of recombinant human hyaluronidase and the second container contains a single fixed dose of rituximab.

Wherein, the concentration of the rituximab can be 10-150 mg/ml; preferably, the concentration is 10-120 mg/ml;

the recombinant human hyaluronidase liquid preparation preferably comprises 45 units/ml to 500000 units/ml of recombinant human hyaluronidase; more preferably, it comprises 45 units/ml to 50000 units/ml of recombinant human hyaluronidase.

Wherein, the dosage of the rituximab is 1400mg or 1600 mg.

Wherein the first container is a vial or a pre-filled needle, preferably a pre-filled needle; the second container is a vial.

In a fifth embodiment, the kit comprises two single-dose containers, wherein the first container contains a single specific dose of said liquid formulation comprising 45 units/ml to 500000 units/ml of recombinant human hyaluronidase, and the second container contains a single fixed dose of trastuzumab.

Wherein the concentration of the trastuzumab is 20-150 mg/ml; preferably 20-120 mg/ml;

the dose of trastuzumab is preferably 600 mg.

The liquid preparation of the recombinant human hyaluronidase preferably comprises 45 units/ml to 100000 units/ml of the recombinant human hyaluronidase; more preferably, it comprises 45 units/ml to 50000 units/ml of recombinant human hyaluronidase.

Wherein the first container is a vial or a pre-filled needle, preferably a pre-filled needle; the second container is a vial.

In a sixth embodiment, the kit comprises two single-dose containers, wherein the first container contains a single specific dose of said liquid formulation comprising 45 units/ml to 500000 units/ml of recombinant human hyaluronidase, and the second container contains a single fixed dose of immunoglobulin.

Wherein the concentration of the immunoglobulin is 5-20% (w/v).

The liquid preparation of the recombinant human hyaluronidase preferably comprises 45 units/ml to 100000 units/ml of the recombinant human hyaluronidase; more preferably, it comprises 45 units/ml to 50000 units/ml of recombinant human hyaluronidase.

Wherein the first container is a vial or a pre-filled needle, preferably a pre-filled needle; the second container is a vial.

In a seventh embodiment, the kit comprises two single-dose containers, wherein the first container contains a single specific dose of said liquid formulation comprising 45 units/ml to 500000 units/ml of recombinant human hyaluronidase, and the second container contains a single fixed dose of daratumab.

Wherein, the concentration of the darunavailability can be 20-120 mg/ml.

The liquid preparation of the recombinant human hyaluronidase preferably comprises 45 units/ml to 100000 units/ml of the recombinant human hyaluronidase; more preferably, it comprises 45 units/ml to 50000 units/ml of recombinant human hyaluronidase.

Wherein, the dosage of the darunavailability is 1800 mg.

Wherein the first container is a vial or a pre-filled needle, preferably a pre-filled needle; the second container is a vial.

In an eighth embodiment, the kit comprises two single-dose containers, wherein the first container contains a single specific dose of said liquid formulation comprising 45 units/ml to 500000 units/ml of recombinant human hyaluronidase, and the second container contains a single fixed dose of human serum albumin.

Wherein the concentration of the human serum albumin is 15-25% (w/v).

The liquid preparation of the recombinant human hyaluronidase preferably comprises 45 units/ml to 100000 units/ml of the recombinant human hyaluronidase; more preferably, it comprises 45 units/ml to 50000 units/ml of recombinant human hyaluronidase.

Wherein the first container is a vial or a pre-filled needle, preferably a pre-filled needle; the second container is a vial.

In a ninth embodiment, the kit comprises two single-dose containers, wherein the first container contains a single specified dose of said liquid formulation comprising 45 units/ml to 500000 units/ml of recombinant human hyaluronidase and the second container contains a single fixed dose of Isatuximab.

Wherein, the concentration of Isatuximab can be 20-120 mg/ml.

The liquid preparation of the recombinant human hyaluronidase preferably comprises 45 units/ml to 100000 units/ml of the recombinant human hyaluronidase; more preferably, it comprises 45 units/ml to 50000 units/ml of recombinant human hyaluronidase.

Wherein the first container is a vial or a pre-filled needle, preferably a pre-filled needle; the second container is a vial.

A third aspect of the invention relates to a method of treating a disease by administering the kit of the second aspect, either separately or in admixture.

The separate administration comprises the following steps:

(a) intradermally or subcutaneously administering the recombinant human hyaluronidase liquid formulation in the kit to a subject;

(b) the subject is then administered the therapeutic agent in the kit.

Wherein steps (a) and (b) may be performed separately, simultaneously or alternately;

when steps (a) and (b) are carried out separately, the time interval between steps (a) and (b) is 0-24 hours;

preferably no time interval, at most 1 minute, at most 2 minutes, at most 3 minutes, at most 4 minutes, at most 5 minutes, at most 6 minutes, at most 7 minutes, at most 8 minutes, at most 9 minutes, at most 10 minutes, at most 15 minutes, at most 20 minutes, at most 25 minutes, at most 30 minutes, at most 1h, at most 2h, at most 3h, at most 6h, at most 12h or at most 24 h.

When administered subcutaneously sequentially, the time interval between steps (a) and (b) may be 0 minutes, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes or 60 minutes.

The separate administration can be achieved by means of a three-way mode, and simultaneous administration or sequential administration can be achieved.

The respective administration can be controlled by an infusion pump or gravity;

wherein the liquid preparation of the recombinant human hyaluronidase can be administered at a rate of 0.1-2 ml/min;

the therapeutic agent may be infused at a rate of 5 ml/hour, 10 ml/hour, 30 ml/hour, 60 ml/hour, 120 ml/hour, 240 ml/hour, or 300 ml/hour.

The mixed administration comprises the following steps: administering subcutaneously to the subject the liquid formulation of recombinant human hyaluronidase in the kit after mixing with a therapeutic agent; the liquid preparation contains 45 to 500000 units of recombinant human hyaluronidase, such as 45 units, 100 units, 150 units, 500 units, 750 units, 1000 units, 2000 units, 3000 units, 4000 units, 5000 units, 6000 units, 7000 units, 8000 units, 9000 units, 10000 units, 15000 units, 20000 units, 25000 units, 30000 units, 35000 units, 40000 units, 45000 units, 50000 units, 55000 units, 60000 units, 65000 units, 70000 units, 75000 units, 80000 units, 85000 units, 90000 units, 95000 units, 100000 units, 105000 units, 110000 units, 115000 units, 120000 units, 125000 units, 130000 units, 135000 units, 140000 units, 145000 units, 150000 units, 155000 units, 160000 units, 165000 units, 180000 units, 185000 units, 190000 units, 195000 units, 300000 units, 170000 units, 250000 units, 400000 units, 250000 units, 400000 units, 20000000 units, 250000 units, 450000 units or 500000 units of recombinant human hyaluronidase.

The mixed administration can be controlled by an infusion pump or gravity;

wherein the liquid preparation of the recombinant human hyaluronidase and the therapeutic agent can be infused at a rate of 5-300 ml/hour, such as 5 ml/hour, 10 ml/hour, 30 ml/hour, 60 ml/hour, 120 ml/hour, 240 ml/hour or 300 ml/hour after mixing.

The therapeutic agent for mixing is in liquid or dry powder form.

Preferably, the enzyme activity of the recombinant human hyaluronidase liquid preparation is 50000 units/ml, and the volume is 0.5-2 ml; when in use, the injection is added into 10-15 ml of 1400mg or 1600mg rituximab injection, and subcutaneous injection is carried out after uniform mixing.

Preferably, the enzyme activity of the recombinant human hyaluronidase liquid preparation is 50000 units/ml, and the volume is 0.25-1 ml; when the injection is used, the injection is added into 4-6 ml of 600mg trastuzumab injection, and subcutaneous injection is performed after uniform mixing.

The recombinant human hyaluronidase liquid preparation can be directly applied and/or applied after dilution.

Wherein the diluting preparation may be conventional in the art, such as physiological saline;

the dilution ratio may be conventional in the art, e.g. 1: 10.

Use of the liquid pharmaceutical composition comprising trastuzumab, pertuzumab and recombinant human hyaluronidase according to any of the above in the preparation of a medicament for treating HER2 positive tumors. In a particular embodiment, the positive tumors include Early Breast Cancer (EBC) or Metastatic Breast Cancer (MBC), gastric cancer, gastroesophageal junction cancer, prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, colon cancer, esophageal cancer, and squamous cell carcinoma of the head and neck. In a preferred embodiment, the positive tumors are breast cancer and gastric cancer.

Wherein the pharmaceutical composition further comprises a chemical agent. The chemical agent is selected from the group consisting of a taxane and an anthracycline. The taxane is paclitaxel (paclitaxel) or docetaxel (docetaxel). The anthracycline can be daunorubicin (daunorubicin), doxorubicin (doxorubicin), or epirubicin (epirubicin).

Use of a pharmaceutical composition comprising rituximab and a liquid preparation of recombinant human hyaluronidase, as described in any of the above, in the preparation of a medicament for the treatment of a CD 20-associated tumor. In a specific embodiment, the tumor comprises relapsed or refractory low grade or follicular, CD20 positive, B cell non-hodgkin lymphoma.

Wherein the pharmaceutical composition further comprises a chemical agent. The chemical is preferably cyclophosphamide, doxorubicin, vincristine or prednisone.

Use of a pharmaceutical composition comprising trastuzumab and a liquid preparation of recombinant human hyaluronidase according to any preceding claim in the preparation of a medicament for treating HER2 positive tumors. In a particular embodiment, the positive tumors include Early Breast Cancer (EBC) or Metastatic Breast Cancer (MBC), gastric cancer, gastroesophageal junction cancer, prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, colon cancer, esophageal cancer, and squamous cell carcinoma of the head and neck. In a preferred embodiment, the positive tumors are breast cancer and gastric cancer.

Wherein the pharmaceutical composition further comprises a chemical agent. The chemical agent is selected from the group consisting of a taxane and an anthracycline. The taxane is paclitaxel (paclitaxel) or docetaxel (docetaxel). The anthracycline can be daunorubicin (daunorubicin), doxorubicin (doxorubicin), or epirubicin (epirubicin).

The use of the pharmaceutical composition comprising the immunoglobulin or recombinant human hyaluronidase liquid preparation as described in any of the above in the preparation of a medicament for the treatment of a disease. In a specific embodiment, the disease is selected from the group consisting of immunodeficiency disease, ANCA-positive systemic necrotizing vasculitis, Common Variant Immunodeficiency Disease (CVID), antibody deficient primary immunodeficiency disease, congenital immunoglobulin deficiency, Wiskott-Aldrich syndrome, X-linked agammaglobulinemia (XLA), Severe Combined Immunodeficiency Disease (SCID), primary hypogammaglobulinemia, infantile hypogammaglobulinemia and antibody-free paraneoplastic cerebellar degenerative alzheimer disease, sepsis, cicatricial pemphigoid, B-cell neoplasm, acquired hypogammaglobulinemia secondary to hematological malignancies, Evans syndrome, acquired hypogammaglobulinemia secondary to hematological malignancies, acute disseminated encephalomyelitis, kawasaki disease, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), multiple sclerosis, autoimmune diseases, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, inflammatory myopathy, high-risk allogeneic hematopoietic stem cell transplantation, IgM paraproteinemia neuropathy, Lambert-Eaton myasthenia syndrome, toxic shock syndrome, multiple myeloma, multifocal motor neuropathy, myasthenia gravis, pemphigus foliaceus, Morsch-Woltmann syndrome, secondary hypogammaglobulinemia, specific antibody deficiency, autoimmune hemolytic anemia, bullous pemphigoid, fetal/neonatal alloimmune thrombocytopenia (FMAIT/NAIT), hemophagocytic syndrome, kidney transplantation, ocular clonic-myoclonic movement disorder, pemphigus vulgaris, post-transfusion purpura, toxic epidermal necrosis/StevenJohnson syndrome (TEN/SJS), systemic lupus erythematosus, inflammatory myopathy, trauma, and bacterial, Viral or fungal infections.

Preferably, the disease is caused by infection with a bacterium, virus or fungus, which may be haemophilus influenzae type B, pseudomonas aeruginosa type a and B, staphylococcus aureus, streptococcus group B, streptococcus pneumoniae (1, 3, 4, 6, 7, 8, 9, 12, 14, 18, 19 and 23), streptococcus pneumoniae type 2 and 5, adenovirus type 2, herpes simplex virus-1, herpes simplex virus-2, cytomegalovirus, epstein-barr virus VCA, hepatitis a virus, hepatitis B virus, influenza a virus, measles virus, parainfluenza virus type 1, 2 and 3, poliovirus in children, varicella-zoster virus, aspergillus or candida albicans.

The use of the pharmaceutical composition comprising the daratumab and the recombinant human hyaluronidase liquid preparation as described in any one of the above for preparing a medicament for treating a disease. In a particular embodiment, the disease is selected from multiple myeloma, light chain amyloidosis, leukemia and lymphoma, solid tumors.

Wherein the pharmaceutical composition further comprises a chemical agent. The chemical agent is selected from glucocorticoids, Proteasome Inhibitors (PIs), cell cycle non-specific drugs, immunomodulating drugs (IMiDs), such as survivin inhibitors, all-trans retinoic acid (ATRA), cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP), dexamethasone, hydrocortisone acetate, bortezomib, melphalan, lenalidomide, thalidomide or pomalidomide.

The use of the pharmaceutical composition comprising human serum albumin and the recombinant human hyaluronidase liquid preparation as described in any of the above in the preparation of a medicament for the treatment of a disease. In a particular embodiment, the disease is selected from shock, cerebral edema due to blood loss trauma and burns, and the treatment of critical conditions such as liver cirrhosis, edema or ascites due to renal disease, and patients with hypoproteinemia.

Use of a pharmaceutical composition comprising Isatuximab, a liquid preparation of recombinant human hyaluronidase as described in any of the above, in the preparation of a medicament for a disease. In a particular embodiment, the disease is selected from multiple myeloma, light chain amyloidosis, leukemia, lymphoma or solid tumors.

Wherein the pharmaceutical composition further comprises a chemical agent. The chemical agent is selected from glucocorticoids, Proteasome Inhibitors (PIs), cell cycle non-specific drugs, immunomodulating drugs (IMiDs), such as survivin inhibitors, all-trans retinoic acid (ATRA), cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP), dexamethasone, hydrocortisone acetate, bortezomib, melphalan, lenalidomide, thalidomide or pomalidomide.

Preferably, the enzyme activity of the recombinant human hyaluronidase liquid preparation is 500 units/ml, and the volume is 0.5-15 ml; when the injection is administered, firstly, the hyaluronidase liquid preparation is subcutaneously injected, and then, the rituximab injection with 1400mg or 1600mg of fixed dose is subcutaneously injected. For patients with various CD20 positive B cell malignancies, the subcutaneous administration can significantly simplify the treatment, changing the original hours of intravenous infusion time to complete injection within 10 minutes, improving the patient treatment experience.

Preferably, the enzyme activity of the recombinant human hyaluronidase liquid preparation is 500 units/ml, and the volume is 0.5-15 ml; when the injection is administered, firstly, the hyaluronidase liquid preparation is subcutaneously injected, and then, the trastuzumab injection with 600mg of fixed dose is subcutaneously injected. For various patients with HER2 positive tumors, the subcutaneous administration can significantly simplify the treatment, changing the original several hours of intravenous infusion time to complete the injection within 10 minutes, improving the patient treatment experience.

The direct administration of the liquid preparation of the recombinant human hyaluronidase can promote the dissipation of local edema or hematoma after operation and trauma, promote the infiltration of local anesthetic, relieve the pain of an injection part or accelerate the absorption of hypodermic or intramuscular injection liquid medicine, relieve the side reaction of hyaluronic acid injection, promote the diffusion of liquid medicine, exudate or blood locally stored in eyes, promote the absorption of vitreous opacity, prevent the adhesion of palpebral bulbus after chemical conjunctival burn and eliminate related inflammatory reaction, improve the reabsorption of a contrast agent barium sulfate (subcutaneous urethrography), and treat traumatic orbital hemorrhage and traumatic retinal edema.

The administration mode for improving reabsorption of the contrast agent barium sulfate is to inject a recombinant human hyaluronidase liquid preparation subcutaneously at the scapular region, wherein the dosage of the liquid preparation is 50-5000 units, and then the contrast agent is injected at the same position.

A fourth aspect of the present invention is directed to packaging materials and injection systems for stable administration according to the third aspect above, the packaging materials including, but not limited to, vials, syringes or test tubes; injection systems include, but are not limited to: a syringe, an infusion pump, an injection pen, a needleless device, or a subcutaneous patch delivery device.

The composition of the injection device may be conventional in the art, including: container, seal, injection needle.

Wherein, the container includes but is not limited to: vials, syringes or test tubes.

The material of the container may be conventional in the art, such as glass or plastic.

Wherein said seal includes, but is not limited to: a sealing plug or a sealing ring.

The material of the sealing element can be conventional in the art, such as rubber, plastic or polymer material.

Wherein, the injection needle head includes but is not limited to water needle, single needle, micro needle group.

The material of the injection needle can be conventional in the art, such as metal, silicon, silica, glass, nickel, titanium or biodegradable polymer.

The water needle includes but is not limited to: a penicillin bottle water needle, an ampoule bottle water needle or a pre-filling injection system.

The ampoule bottle water needle can be a glass ampoule or a plastic ampoule.

The pre-filled injection system may be conventional in the art, such as a pre-filled syringe.

Preferably, the container is a penicillin bottle made of neutral borosilicate glass and has the specification of 0.1-20 ml;

the sealing element is a sealing plug and is made of halogenated butyl rubber;

the injection needle head is a single needle or a micro-needle group.

Preferably, the material of the single needle can be 304 or 316 stainless steel, and the specification (as shown in table 1) can be 30G, 24G, 27G and 29G; the micro needle group can be made of 304 or 316 stainless steel and biodegradable polymer, and is a nano-scale needle with the height of 10-2000 mu m and the width of 10-50 mu m.

TABLE 1 Single needle Specification

Specification of	Inner diameter
		14G	1.54mm
15G	1.36mm
		16G	1.19mm
18G	0.84mm
		20G	0.60mm
21G	0.51mm
		22G	0.41mm
23G	0.34mm
		24G	0.30mm
25G	0.26mm
		26G	0.23mm
27G	0.21mm
		29G	0.18mm
30G	0.16mm
		32G	0.11mm
34G	0.06mm

In one embodiment of the invention, an article of manufacture is provided containing a pharmaceutical formulation according to the second aspect of the invention and instructions for its use are provided. This article comprises the container. The article of manufacture may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, infusion pumps, and package inserts printed with instructions for use.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the stability of the recombinant human hyaluronidase at 2-8 ℃ can be maintained without special additives such as human serum albumin and the like which have infection risks, and the stability is kept above 95% after the recombinant human hyaluronidase is placed for 12 months. The preparation can be administered alone, simultaneously or sequentially with other preparations, or mixed with other preparations. The clinical use is more convenient than the freeze-drying preparation; can improve the treatment experience of patients and reduce the side effect related to infusion.

Drawings

FIG. 1 is a schematic diagram of the stability study of the liquid formulations of recombinant human hyaluronidase in different buffers.

FIG. 2 is a schematic diagram of the stability studies of liquid formulations of recombinant human hyaluronidase at various methionine concentrations.

FIG. 3 is a schematic diagram of the study of the stability of the recombinant human hyaluronidase liquid formulations with different concentrations of sodium chloride, trehalose, and sucrose.

FIG. 4 is a schematic diagram of the stability study of the recombinant human hyaluronidase liquid formulations at different surfactant concentrations.

FIG. 5 is a schematic diagram of the stability study of the liquid formulations of recombinant human hyaluronidase at different concentrations.

Figure 6 is a schematic of a hyaluronidase liquid formulation stability study with one stabilizer reduced.

FIG. 7 is a pathological image of brain tissue, heart tissue and liver tissue in example 6;

wherein: (a) brain tissue of a normal saline control group, (b) brain tissue of a high concentration PH20 preparation group, (c) heart tissue of a normal saline control group, (d) heart tissue of a high concentration PH20 preparation group, (e) liver tissue of a normal saline control group, and (f) liver tissue of a high concentration PH20 preparation group.

FIG. 8 is a histopathological image of the major organs of example 6;

wherein: (a) spleen tissue of a normal saline control group, (b) spleen tissue of a high-concentration PH20 preparation group, (c) lung tissue of a normal saline control group, (d) lung tissue of a high-concentration PH20 preparation group, (e) kidney tissue of a normal saline control group, and (f) kidney tissue of a high-concentration PH20 preparation group.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1 preparation of human hyaluronidase protein

CHO cells are adopted to be cultured in a serum-free culture medium which is independently developed in a suspension way, the serum-free feed medium which is independently developed is used for feeding and feeding control culture, and the scale of the reactor with the volume of 30L is gradually enlarged through shaking culture.

When the culture is carried out for 3-4 days, the amount of the supplemented culture medium added into the bioreactor every day is 2% -5% of the actual culture volume in the bioreactor. The culture temperature is controlled between 35 ℃ and 37 ℃, and 10 percent of Na is supplemented₂CO₃And CO₂Controlling the pH value to be 7.0; controlling the aeration quantity of the reactor to be 0.015-0.15 vvm; the rotating speed is controlled to be 80-150 rpm; the dissolved oxygen value is controlled to be 20-40%. Sampling every day during the cell culture process, and monitoring temperature, pH, glucose concentration, lactic acid concentration, osmolality and protein expression; and (3) finishing the culture when the CHO cell survival rate is lower than 80% or the culture period reaches 14-20 days, and obtaining the recombinant human hyaluronidase.

And sequentially carrying out deep filtration, anion chromatography, affinity chromatography, hydrophobic chromatography, cation chromatography and ultrafiltration liquid exchange on the obtained recombinant human hyaluronidase supernatant. And (3) producing three batches of stock solutions according to the process to obtain the recombinant human hyaluronidase stock solution with SEC purity of more than 95%, the hyaluronidase with RP purity of more than 85%, and the enzyme activity of more than 70000 units/mg.

Example 2 recombinant human hyaluronidase liquid formulation study

The stock solution of recombinant human hyaluronidase obtained in example 1 was changed to liquid formulations of different compositions, and the concentration of recombinant human hyaluronidase was adjusted to the desired concentration. All liquid formulations were sterile filtered through a 0.22 μm low protein adsorption filter and filled aseptically into sterile 5ml glass vials, stoppered with fluororesin-laminated butyl rubber stoppers and capped with aluminum/plastic flip-off seals. The fill volume was 2 ml. The formulations were stored at different temperatures and sampled at specified time intervals for formulation stability and the stability data were compiled for different liquid formulations. Samples were taken at 25 ℃ for accelerated experiments and at different times the protein quality was analyzed by the following analytical method. The analytical methods included SEC purity, RP purity, enzyme activity, and the results are shown in FIGS. 1-6.

As shown in fig. 1, the SEC purity and enzyme activity decreased significantly after the recombinant human hyaluronidase at pH 4.5 and pH 8.5 was left at 25 ℃ for a period of time; the buffer is 5mM and 50mM phosphate buffer solution, histidine buffer solution, acetate buffer solution, citrate buffer solution and Tris buffer solution, and the recombinant human hyaluronidase liquid preparation under the conditions of pH5.0, 6.5 and 8.0 has better stability.

As shown in FIG. 2, the RP purity and enzyme activity decreased significantly after the recombinant human hyaluronidase was placed at 25 ℃ for a period of time at a concentration of 2mM methionine; the recombinant human hyaluronidase liquid formulations containing methionine concentrations in the range of 5, 10 and 50mM had better stability.

As shown in fig. 3, the reduction of SEC purity and enzyme activity was significant after the recombinant human hyaluronidase in the presence of 20mM sodium chloride, 250mM trehalose and 20mM sodium chloride, 250mM sucrose stabilizer was left at 25 ℃ for a period of time; the stability of the recombinant human hyaluronidase liquid preparation containing the sodium chloride with the concentration of 30mM, 130mM and 180mM, the trehalose with the concentration of 25mM, 53mM and 200mM and the sucrose with the concentration of 25mM, 53mM and 200mM is better.

As shown in figure 4, the recombinant human hyaluronidase liquid preparation containing 0.01-0.1% of polysorbate 20, 0.01-0.1% of polysorbate 80 and 0.01-0.1% of poloxamer 188 has good stability.

As shown in fig. 5, the recombinant human hyaluronidase activity in the recombinant human hyaluronidase liquid preparation of this example is better in stability at 150, 500, 1000, 5000, 50000, 300000, 1500000, 3000000 and 4500000 units/ml, and the enzyme activity is maintained at 95% or more after 12 months of storage at 2-8 ℃.

As shown in fig. 6, the more components in a protein drug formulation, the greater the risk of uncontrollable. The stability research of the hyaluronidase liquid preparation without one stabilizer shows that the carbohydrate stabilizer is removed from the recombinant human hyaluronidase liquid preparation, the stability of the recombinant human hyaluronidase liquid preparation with different concentrations is still good, and the enzyme activity of each preparation is kept above 95% after the preparation is placed at 2-8 ℃ for 12 months.

Example 3 recombinant human Hyaluronidase liquid formulation increases the absorption rate of high concentration protein solution subcutaneously infused into nude mice

The experiment aims to observe the influence of subcutaneous injection of the hyaluronidase liquid preparation on subcutaneous pressure infusion of high-concentration human serum albumin solution on the back of a nude mouse, record the infusion time and calculate the infusion rate.

36 female BALB/c nude mice, randomly divided into 2 groups by weight: 1-positive control group (50 units/spot) and 2-hyaluronidase liquid formulation group (50 units/spot), each group containing 18 animals. After animals were anesthetized, 2 injection points were symmetrically drawn on the back with a MARK pen, and then hyaluronidase liquid preparation or positive control solution was administered by subcutaneous injection on the right back, and a corresponding volume of physiological saline was administered by subcutaneous injection on the left back, as an autologous negative control. A1 mL syringe containing 50mg/mL of human serum albumin solution is connected to one end of a PE tube with the inner diameter of 0.76mm, a 0.55mm needle is connected to the other end of the PE tube, 600 uL of human serum albumin is subcutaneously infused at the administration site under the water pressure of 20cm, 30cm and 40cm (6 animals per water pressure in each group), the infusion time t (min) is recorded, and the infusion rate v (uL/min) is calculated.

The infusion speeds of the human blood albumin solution under the subcutaneous conditions (given physiological saline) of the left back of the animals in the hyaluronidase liquid preparation group under the water pressure of 20cm, 30cm and 40cm are respectively 54.9 +/-6.8 mu L/min, 113.0 +/-20.1 mu L/min and 201.5 +/-48.3 mu L/min; the transfusion speeds of human blood albumin solution under different water pressures of the subcutaneous part of the right back (given the hyaluronidase liquid preparation) are 326.1 +/-38.4 mu L/min, 749.3 +/-40.6 mu L/min and 1140.5 +/-160.4 mu L/min respectively, and compared with the left self-negative control group, the transfusion speeds of the human blood albumin under different pressures are obviously increased (P is less than or equal to 0.01).

Under the experimental condition, the hyaluronidase liquid preparation can obviously increase the speed of subcutaneous infusion of high-concentration human serum albumin solution into nude mice.

Example 4 recombinant human hyaluronidase liquid formulation increases the rate of absorption of subcutaneous rituximab solution in nude mice

The experiment aims to observe the influence of subcutaneous injection of the hyaluronidase liquid preparation on subcutaneous pressure infusion of a high-concentration rituximab (120mg/ml) solution at the back of a nude mouse, record the infusion time and calculate the infusion rate.

36 female BALB/c nude mice, randomly divided into 3 groups by body weight: 1-negative control group (normal saline + rituximab), 2-hyaluronidase liquid preparation group (50 units/point are firstly administrated and then the monoclonal antibody is injected), 3-hyaluronidase liquid preparation group + rituximab mixed group, and each group has 12 animals. In group 3, before administration, 300000 units/ml of recombinant human hyaluronic acid solution was added to the rituximab solution at a ratio of 1:60, and administration was performed after mixing well. After animals were anesthetized, 2 injection points were symmetrically drawn on the back with a MARK pen, and then hyaluronidase liquid preparation or positive control solution was administered by subcutaneous injection on the right back, and a corresponding volume of physiological saline was administered by subcutaneous injection on the left back, as an autologous negative control. A1 mL syringe containing 120mg/mL rituximab solution is connected to one end of a PE tube with the inner diameter of 0.76mm, a 0.55mm needle is connected to the other end of the PE tube, 600 mu L of human rituximab solution is subcutaneously infused at the administration position under the water pressure of 20cm, 30cm and 40cm (6 animals per water pressure in each group), the infusion time t (min) is recorded, and the infusion rate v (mu L/min) is calculated.

The left side and back subcutaneous (given physiological saline) injection speeds of rituximab solutions under water pressures of 20cm, 30cm and 40cm in the hyaluronidase liquid preparation group animals are 44.5 +/-3.4 mu L/min, 92.1 +/-15.2 mu L/min and 182.5 +/-38.1 mu L/min respectively; the infusion speeds of the rituximab solution under different water pressures of the right dorsal subcutaneous side (given the hyaluronidase liquid preparation) are 305.1 +/-39.5 mu L/min, 734.2 +/-51.2 mu L/min and 1251.3 +/-171.7 mu L/min respectively, and compared with the left autologous negative control group, the infusion speeds of the rituximab solution under different pressures are obviously increased (P is less than or equal to 0.01).

Under the experimental condition, the hyaluronidase liquid preparation can obviously increase the speed of subcutaneous infusion of high-concentration rituximab solution in nude mice.

Example 5 recombinant human Hyaluronidase liquid formulation increases absorption Rate of trastuzumab solution subcutaneously infused in nude mice

The experiment aims to observe the influence of subcutaneous injection of the hyaluronidase liquid preparation on subcutaneous pressure infusion of a high-concentration trastuzumab (120mg/ml) solution at the back of a nude mouse, record the infusion time and calculate the infusion rate.

36 female BALB/c nude mice, randomly divided into 3 groups by body weight: 1-negative control group (normal saline + trastuzumab), 2-hyaluronidase liquid preparation group (50 units/point are administered first and then monoclonal antibody is injected), 3-hyaluronidase liquid preparation + trastuzumab mixed group, and each group contains 12 animals. In group 3, before administration, 300000 units/ml of recombinant human hyaluronic acid solution was added to trastuzumab solution at a ratio of 1:60, and administration was performed after mixing well. After animals were anesthetized, 2 injection points were symmetrically drawn on the back with a MARK pen, and then hyaluronidase liquid preparation or positive control solution was administered by subcutaneous injection on the right back, and a corresponding volume of physiological saline was administered by subcutaneous injection on the left back, as an autologous negative control. A1 mL syringe containing 120mg/mL trastuzumab solution is connected to one end of a PE tube with the inner diameter of 0.76mm, a 0.55mm needle is connected to the other end of the PE tube, 600 mu L of human trastuzumab solution is subcutaneously infused at the administration position under the water pressure of 20cm, 30cm and 40cm respectively (6 animals per water pressure in each group), the infusion time t (min) is recorded, and the infusion rate v (mu L/min) is calculated.

The infusion rates of trastuzumab solution under water pressure of 20cm, 30cm and 40cm under left side and back subcutaneous (given physiological saline) of the hyaluronidase liquid preparation group animals are 34.5 +/-7.4 mu L/min, 111.8 +/-19.6 mu L/min and 192.5 +/-54.5 mu L/min respectively; the infusion speeds of trastuzumab solution under different water pressures of the subcutaneous part of the right back (given with the hyaluronidase liquid preparation) are 284.4 +/-52.4 mu L/min, 851.2 +/-60.3 mu L/min and 1004.1 +/-187.1 mu L/min respectively, and compared with the left self-negative control group, the infusion speeds of the trastuzumab solution under different pressures are obviously increased (P is less than or equal to 0.01).

Under the experimental condition, the hyaluronidase liquid preparation can obviously increase the speed of subcutaneous infusion of high-concentration trastuzumab solution in nude mice.

Example 6 evaluation of safety of high concentration liquid preparation of recombinant human hyaluronidase

A toxicity test of repeated subcutaneous injections was performed on cynomolgus monkeys to evaluate the safety of the high concentration PH20 formulation.

20 cynomolgus monkeys were divided by body weight into a normal saline control group and a high concentration PH20 preparation group, each group having 5 animals/sex. The high-concentration PH20 preparation was administered to animals at a dose of 0.04mg/kg (equivalent to 12000 units/kg) 1 time per day for 28 consecutive days. The dose of each animal was determined according to the weight of the prodrug and administered by subcutaneous injection on the inner side of both hind limbs. The saline control group animals were given daily subcutaneous injections of saline, and the remaining experimental conditions were the same as those of the high concentration PH20 preparation group animals.

All animals were not moribund or dead during the dosing period, and all animals were euthanized the next day after the end of the dosing period (D29), dissected systemically, and examined histopathologically for major organs. The results show that the animal gross anatomy and microscopic examination results of the normal saline control group and the high-concentration PH20 preparation group have no abnormal pathological changes related to injection administration, and the histopathological pictures of the main organs are shown in figures 7-8.

All animals were observed 1 time daily before and after the last dose during the administration period, and the injection site was observed for changes such as swelling, fever, edema, erythema, ulceration, etc. The results show that the local clinical observation of all animals is not abnormal during the administration period; the local gross anatomy of all animals administered the day after the last dose (D29) showed no abnormal pathological changes associated with the administration by injection; no irritation associated with the high concentration of the pH20 formulation was observed microscopically at the injection site in the animals in the high concentration of the pH20 formulation as compared to the saline control.

Furthermore, there was no significant abnormality in the hematological indices of all animals compared to the control group, which was exemplified by the hematological index on the day following the last dose (D29), and WBC (x 10) in the saline control group and the high concentration PH20 formulation group⁹/L) was 9.356. + -. 1.615 and 9.174. + -. 1.569, respectively, and Neut (%) was 31.92. + -. 7.52 and 37.70. + -. 14, respectively.89, Lymph (%) 58.94. + -. 7.08 and 55.06. + -. 15.13, RBC (. times.10)¹²/L) 5.654 + -0.295 and 5.368 + -0.364, HGB (g/L) 128.4 + -6.7 and 123.2 + -6.0, PLT (. times.10)⁹/L) are 341.8 +/-97.5, 346.8 +/-62.3 and the like respectively.

In terms of biochemical indexes of animal blood, the physiological saline control group and the high concentration PH20 preparation group have no abnormal change, taking the biochemical indexes of the next day after the last drug (D29) as an example, the physiological saline control group and the high concentration PH20 preparation group have ALT (U/L) of 33.5 + -4.9 and 35.6 + -5.1, AST (U/L) of 63.4 + -17.9 and 68.0 + -6.3, ALP (U/L) of 405.6 + -81.7 and 405.6 + -89.5, TBil (mu mol/L) of 2.268 + -0.516 and 2.182 + -0.698, LDH (U/L) of 637.4 + -131.9 and 2 + -111.5, Cre (mu mol/L) of 61.6 + -10.7 and 62.6 + -9.5, TG (mmol/L) of 0.080.9 and 468.080 + -111.5, respectively, Glu (mu mol/L) of 468.6 + -10.7 and 62.6 + -9.5, TG (TG/L) of 0.74 + -0.74 g + -69 and 38.64 g + -3.64 g, respectively, and the like.

Thus, the high concentration PH20 formulation is safe for subcutaneous injection and non-irritating to the topical application.

SEQUENCE LISTING

<110> Shanghai Baoji pharmaceutical Co., Ltd

<120> liquid preparation of recombinant human hyaluronidase and application thereof

<130> P20013061C

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 442

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of recombinant human hyaluronidase

<400> 1

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Leu Ser Ile Met Arg Ser Met Lys Ser Cys Leu Leu Leu Asp

305 310 315 320

Asn Tyr Met Glu Thr Ile Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu

435 440

<210> 2

<211> 447

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of PH20

<400> 2

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Leu Ser Ile Met Arg Ser Met Lys Ser Cys Leu Leu Leu Asp

305 310 315 320

Asn Tyr Met Glu Thr Ile Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr

435 440 445