CN114762677A

CN114762677A - Recombinant human hyaluronidase preparation and application thereof

Info

Publication number: CN114762677A
Application number: CN202011612733.2A
Authority: CN
Inventors: 王征; 刘彦君
Original assignee: Shanghai Baoji Pharmaceutical Co ltd
Current assignee: Shanghai Baoji Pharmaceutical Co ltd
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2022-07-19

Abstract

The invention discloses a recombinant human hyaluronidase preparation and application thereof, wherein the recombinant human hyaluronidase preparation comprises; 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 recombinant human hyaluronidase preparation can be applied to preparation of subcutaneous infusion drugs. The auxiliary subcutaneous infusion drug can ensure that the large infusion drug which can only be administrated through veins is administrated in a subcutaneous infusion mode, and the clinical use is more convenient, safe and comfortable.

Description

Recombinant human hyaluronidase preparation and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a recombinant human hyaluronidase preparation and application thereof in preparation of a medicine for assisting subcutaneous infusion.

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 the drug is limited, the rate of drug arrival at the blood vessel is reduced, and the volume of liquid injected subcutaneously is limited.

Hyaluronidase (HAase), 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, placenta, and the like. 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 freeze-dried hyaluronidase in a crude state contains a large amount of impurities, has low purity, and has poor stability when prepared into a liquid formulation.

Intravenous infusion refers to a method of infusing a large amount of sterile liquid, electrolytes and drugs into the body through veins. Although the medicine can be quickly and directly administered to patients to enable the medicine to reach the therapeutic concentration, the intravenous infusion has obvious adverse reaction. The adverse reactions of the intravenous infusion mainly comprise adverse drug reactions, needle sickness, phlebitis, heavy circulation load, air embolism, infection and the like, the pain of a patient is increased, the treatment is hindered, and the serious adverse infusion reactions even can endanger the life. According to incomplete statistics, the transfusion quantity of China is more than one billion bottles every year, and about 20 ten thousand people die of adverse reactions of transfusion every year. The average infusion amount of each person is 2.5-3.3 bottles per year internationally, and 8 bottles are used in China. Since 2015, regulations for limiting and regulating intravenous infusion therapy have been issued by a number of provinces in order to reduce the occurrence of therapeutic accidents of intravenous infusion.

Therefore, in order to facilitate clinical use and reduce risks, a safe, effective and comfortable infusion mode is urgently needed to replace the traditional intravenous infusion.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the prior art and provide the application of the recombinant human hyaluronidase preparation in preparing subcutaneous infusion medicines, and the preparation has the advantages of convenient clinical use, wide application, simple preparation formula, good product stability, low irritation, safety and effectiveness. Can solve the problem that the large-volume transfusion is difficult to be carried out by a subcutaneous administration route and the transfusion is required to be carried out by a vein during the clinical treatment. When the recombinant human hyaluronidase-assisted drug subcutaneous infusion animal experiment is carried out, the invention unexpectedly discovers that the drug liquid can be rapidly diffused and absorbed after being injected into the subcutaneous space of a small pig by combining with the hyaluronidase, compared with a negative control group (which is not combined with the hyaluronidase, and directly infuses the drug liquid subcutaneously), the experimental group can significantly reduce the subcutaneous infusion pressure of the drug liquid, reduce the swelling and deformation degree of the skin after infusion and the time required for recovering to be normal, the infusion part has no obvious red and swollen, and the rapid diffusion and absorption of the infused drug liquid are assisted, so that the subcutaneous administration of the drug liquid with large volume becomes possible.

The invention solves the technical problems through the following technical scheme:

1. hyaluronidase preparation and use

A first aspect of the invention relates to a recombinant human hyaluronidase formulation 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 5-50 mM;

the concentration of the stabilizer is 1-500 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 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 8.0, 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 provided by the histidine buffer is 5.5-7.5, the pH provided by the acetic acid buffer is 5.0-6.0, the pH provided by the phosphate buffer is 6.0-8.0, the pH provided by the citric acid buffer is 5.0-7.0, and the pH provided by the Tris buffer is 7.0-8.0.

Preferably, the pH of the histidine buffer is 6.0.

Preferably, the acetic buffer has a pH of 5.0.

Preferably, the phosphate buffer has a pH of 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 present 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 mannitol, sucrose, trehalose, sodium chloride and methionine; for example, the stabilizer contains methionine and two or more selected from the group consisting of trehalose, sucrose, mannitol, and sodium chloride.

The concentration of the stabilizer is preferably 100 to 400mM, preferably 150 to 350mM, for example, 155mM, 188mM, 193mM, 215mM, 233mM, 240mM, 263mM, 283mM, 292mM, 316mM or 330 mM. A

Preferably, the trehalose concentration is 25-200 mM, such as 25mM, 53mM, 132mM or 200 mM.

Preferably, the sucrose concentration is 20 to 200mM, such as 25mM, 53mM, 132mM or 200 mM. Preferably, the sodium chloride concentration is 30-250 mM, such as 30mM, 50mM, 90mM, 130mM, 145mM, 170mM or 250 mM.

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

Preferably, the concentration of mannitol is 150-300 mM, such as 165mM, 220mM or 280 mM.

The recombinant human hyaluronidase preparation can be used as a liquid preparation, and can also be subjected to freeze-drying treatment to prepare a freeze-dried preparation. In a preferred embodiment of the present invention, the concentration of the stabilizer is 185-380 mM, and the stabilizer at least comprises mannitol, methionine, and trehalose or sucrose; wherein the concentration of the mannitol is 150-280 mM, the concentration of the methionine is 5-50 mM, and the concentration of the trehalose or sucrose is 25-130 mM. The formulation of this embodiment is more suitable for preparation as a lyophilized formulation.

The nonionic surfactant is selected from one or more of 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 recombinant human hyaluronidase preparation is one of the following compositions:

in a specific embodiment of the present invention, the stabilizer of the preparation of recombinant human hyaluronidase is one of the following compositions:

(1)145mM sodium chloride and 10mM methionine;

(2)130mM sodium chloride, 53mM trehalose and 10mM methionine;

(3)130mM sodium chloride, 53mM sucrose and 10mM methionine;

(4)26mM trehalose, 280mM mannitol and 10mM methionine.

(5)30mM sodium chloride, 200mM trehalose and 10mM methionine;

(6)180mM sodium chloride, 25mM trehalose and 10mM methionine;

(7)30mM sodium chloride, 200mM sucrose and 10mM methionine;

(8)180mM sodium chloride, 25mM sucrose and 10mM methionine;

(9)130mM sodium chloride, 53mM trehalose and 5mM methionine;

(10)130mM sodium chloride, 53mM trehalose and 50mM methionine;

(11)130mM sodium chloride, 53mM trehalose and 10mM methionine;

(12)22mM mannitol, 53mM trehalose and 10mM methionine;

(13)53mM trehalose, 220mM mannitol and 10mM methionine;

(14)53mM trehalose, 220mM mannitol and 5mM methionine;

(15)53mM trehalose, 220mM mannitol and 50mM methionine;

(16)150mM mannitol, 170mM sodium chloride and 10mM methionine;

(17)220mM mannitol, 53mM trehalose and 10mM methionine;

(18)150mM mannitol, 50mM sodium chloride, 53mM trehalose and 10mM methionine;

(19)150mM mannitol, 90mM sodium chloride, 130mM trehalose and 10mM methionine;

(20)150mM mannitol, 25mM trehalose and 10mM methionine;

(21)220mM mannitol, 53mM sucrose and 10mM methionine;

(22)150mM mannitol, 50mM sodium chloride, 53mM sucrose and 10mM methionine;

(23)150mM mannitol, 90mM sodium chloride, 130mM sucrose and 10mM methionine;

(24)150mM mannitol, 25mM sucrose and 10mM methionine;

(25)132mM trehalose, 150mM mannitol and 10mM methionine.

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

Preferably, in the present invention, the recombinant hyaluronidase is purified from CHO-expressed cell supernatant.

Preferably, the purity of the recombinant hyaluronidase is more than 95%, and the specific activity is more than 70000 units/mg.

(1)5000 units/ml recombinant human hyaluronidase, 5mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 20, pH 7.0;

(2)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 20, pH 7.0;

(3)5000 units/ml recombinant human hyaluronidase, 50mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 20, pH 7.0;

(4)5000 units/ml recombinant human hyaluronidase, 5mM histidine buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 20, pH 6.0;

(5)5000 units/ml recombinant human hyaluronidase, 50mM histidine buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 20, pH 6.0;

(6)5000 units/ml recombinant human hyaluronidase, 5mM acetate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 20, pH 5.0;

(7)5000 units/ml recombinant human hyaluronidase, 50mM acetate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 20, pH 5.0;

(8)5000 units/ml recombinant human hyaluronidase, 5mM citrate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 20, pH 6.5;

(9)5000 units/ml recombinant human hyaluronidase, 50mM citrate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 20, pH 6.5;

(10)5000 units/ml recombinant human hyaluronidase, 5mM Tris buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 20, pH 8.0;

(11)5000 units/ml recombinant human hyaluronidase, 50mM Tris buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 20, pH 8.0;

(12)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 5mM methionine and 0.02% (w/v) polysorbate 20, pH 7.0;

(13)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 50mM methionine and 0.02% (w/v) polysorbate 20, pH 7.0;

(14)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 200mM trehalose, 30mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 20, pH 7.0;

(15)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02% (w/v) poloxamer 188, pH 7.0;

(16)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 25mM trehalose, 180mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 20, pH 7.0;

(17)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 200mM sucrose, 30mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 20, pH 7.0;

(18)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM sucrose, 130mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 20, pH 7.0;

(19)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 25mM sucrose, 180mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 20, pH 7.0;

(20)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02% (w/v) polysorbate 80, pH 7.0;

(21)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.01% (w/v) polysorbate 80, pH 7.0;

(22)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.10% (w/v) polysorbate 80, pH 7.0;

(23)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.01% (w/v) polysorbate 20, pH 7.0;

(24)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.10% (w/v) polysorbate 20, pH 7.0;

(25)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.01% (w/v) poloxamer 188, pH 7.0;

(26)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.10% (w/v) poloxamer 188, pH 7.0;

(27)500 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02(w/v) polysorbate 20, pH 7.0;

(28)45 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02(w/v) polysorbate 20, pH 7.0;

(29)150 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02(w/v) polysorbate 20, pH 7.0;

(30)1000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02(w/v) polysorbate 20, pH 7.0;

(31)50000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02(w/v) polysorbate 20, pH 7.0;

(32)300000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02(w/v) polysorbate 20, pH 7.0;

(33)1500000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02(w/v) polysorbate 20, pH 7.0;

(34)3000000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02(w/v) polysorbate 20, pH 7.0;

(35)4500000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 130mM sodium chloride, 10mM methionine and 0.02(w/v) polysorbate 20, pH 7.0;

(36)4500000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 145mM sodium chloride, 10mM methionine and 0.02(w/v) polysorbate 20, pH 7.0;

(37)1500000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 145mM sodium chloride, 10mM methionine and 0.02(w/v) polysorbate 20, pH 7.0;

(38)300000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 145mM sodium chloride, 10mM methionine and 0.02(w/v) polysorbate 20, pH 7.0;

(39)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 145mM sodium chloride, 10mM methionine and 0.02(w/v) polysorbate 20, pH 7.0;

(40)150 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 145mM sodium chloride, 10mM methionine and 0.02(w/v) polysorbate 20, pH 7.0;

(41)45 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 145mM sodium chloride, 10mM methionine and 0.02(w/v) polysorbate 20, pH 7.0;

(42)45 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 22mM mannitol, 53mM trehalose, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(43)500 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 22mM mannitol, 53mM trehalose, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(44)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 22mM mannitol, 53mM trehalose, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(45)50000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 22mM mannitol, 53mM trehalose, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(46)300000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 22mM mannitol, 53mM trehalose, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(47)4500000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 22mM mannitol, 53mM trehalose, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(48)5000 units/ml recombinant human hyaluronidase, 5mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(49)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(50)5000 units/ml recombinant human hyaluronidase, 50mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(51)5000 units/ml recombinant human hyaluronidase, 5mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 6.0;

(52)5000 units/ml recombinant human hyaluronidase, 50mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 6.0;

(53)5000 units/ml recombinant human hyaluronidase, 50mM acetate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 5.0;

(54)5000 units/ml recombinant human hyaluronidase, 5mM acetate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 5.0;

(55)5000 units/ml recombinant human hyaluronidase, 50mM citrate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 6.5;

(56)5000 units/ml recombinant human hyaluronidase, 5mM citrate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 6.5;

(57)5000 units/ml recombinant human hyaluronidase, 50mM Tris buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 8.0;

(58)5000 units/ml recombinant human hyaluronidase, 5mM Tris buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 8.0;

(59)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 50mM methionine and 0.02% polysorbate 20, pH 7.0;

(60)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 5mM methionine and 0.02% polysorbate 20, pH 7.0;

(61)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 150mM mannitol, 170mM sodium chloride, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(62)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 220mM mannitol, 53mM trehalose, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(63)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 150mM mannitol, 50mM sodium chloride, 53mM trehalose, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(64)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 150mM mannitol, 90mM sodium chloride, 130mM trehalose, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(65)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 150mM mannitol, 25mM trehalose, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(66)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 220mM mannitol, 53mM sucrose, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(67)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 150mM mannitol, 50mM sodium chloride, 53mM sucrose, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(68)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 150mM mannitol, 90mM sodium chloride, 130mM sucrose, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(69)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 150mM mannitol, 25mM sucrose, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(70)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 132mM trehalose, 150mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(71)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 26mM trehalose, 280mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 7.0.

(72)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.01% polysorbate 20, pH 7.0;

(73)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.10% polysorbate 20, pH 7.0;

(74)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.01% polysorbate 80, pH 7.0;

(75)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.10% polysorbate 80, pH 7.0;

(76)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 80, pH 7.0;

(77)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.10% poloxamer 188, pH 7.0;

(78)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.01% poloxamer 188, pH 7.0;

(79)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% poloxamer 188, pH 7.0;

(80)150 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(81)500 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(82)1000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(83)300000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(84)50000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(85)1500000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(86)3000000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

(87)4500000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 53mM trehalose, 220mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 7.0.

As described above, the preparation of the recombinant hyaluronidase of the present invention can be prepared in a lyophilized form.

Preferably, the cryoprotectant is any one or a combination of at least two of sucrose, trehalose, maltose or lactose, preferably sucrose and/or trehalose. Most preferably 20g/L trehalose.

Preferably, the excipient is mannitol and/or glycine, preferably mannitol. Most preferably 40g/L mannitol.

Preferably, the lyophilized preparation is dissolved in water for injection and then used for subcutaneous injection.

In another aspect, the present invention provides a method for preparing a lyophilized formulation of recombinant hyaluronidase according to the first aspect, the method comprising the steps of:

A. adding the recombinant hyaluronidase, a buffering agent, a freezing protective agent, an excipient, an enzyme activity protective agent and a surfactant into water, and uniformly mixing to prepare a recombinant hyaluronidase liquid preparation;

B. and D, freeze-drying the recombinant hyaluronidase liquid preparation obtained in the step A to obtain a recombinant hyaluronidase freeze-dried preparation.

Preferably, the freeze-drying process is:

(1) pre-freezing at-2 deg.C to-8 deg.C, such as-2 deg.C, -3 deg.C, -4 deg.C, -5 deg.C, -6 deg.C, -7 deg.C or-8 deg.C, for 0.5-1 h, such as 0.5h, 0.6h, 0.7h, 0.8h, 0.9h or 1 h;

(2) pre-freezing for 2-4 h, such as 2h, 2.2h, 2.4h, 2.6h, 2.8h, 3h, 3.3h, 3.5h, 3.7h, 3.9h or 4h at-35-40 ℃ environment, such as-35 ℃, -36 ℃, -37 ℃, -38 ℃, -39 ℃ or-40 ℃;

(3) drying for 15-25 h, such as 15h, 16h, 17h, 18h, 19h, 20h, 21h, 23h, 24h or 25h in an environment of-20 ℃ to-30 ℃, such as-20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃ or-30 ℃, 10-20 mbar, such as 10mbar, 11mbar, 12mbar, 13mbar, 14mbar, 15mbar, 16mbar, 17mbar, 18mbar, 19mbar or20 mbar;

(4) drying at-15 deg.C to-25 deg.C, such as-15 deg.C, -16 deg.C, -17 deg.C, -18 deg.C, -19 deg.C, -20 deg.C, -21 deg.C, -22 deg.C, -23 deg.C, -24 deg.C or-25 deg.C, such as 0.1mbar, 0.11mbar, 0.12mbar, 0.13mbar, 0.14mbar, 0.15mbar, 0.16mbar, 0.17mbar, 0.18mbar, 0.19mbar or 0.2mbar for 0.5 to 3 hours, such as 0.5h, 0.7h, 0.9h, 1h, 1.3h, 1.5h, 1.8h, 2h, 2.2h, 2.4h, 2.6h, 2.8h or 3 h;

(5) drying at-5 deg.C to 10 deg.C, such as-5 deg.C, -4 deg.C, -3 deg.C, -2 deg.C, -1 deg.C, 0 deg.C, 1 deg.C, 2 deg.C, 3 deg.C, 4 deg.C, 5 deg.C, 6 deg.C, 7 deg.C, 8 deg.C, 9 deg.C or 10 deg.C, such as 0.1mbar, 0.11mbar, 0.12mbar, 0.13mbar, 0.14mbar, 0.15mbar, 0.16mbar, 0.17mbar, 0.18mbar, 0.19mbar or 0.2mbar for 0.5-3 h, such as 0.5h, 0.7h, 0.9h, 1h, 1.3h, 1.5h, 1.8h, 2h, 2.2h, 2.4h, 2.6h, 2.8h or 3 h;

(6) drying for 5-15 h, such as 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h or 15h, in an environment of 15-30 deg.C, such as 15 deg.C, 16 deg.C, 17 deg.C, 18 deg.C, 19 deg.C, 20 deg.C, 22 deg.C, 24 deg.C, 26 deg.C, 28 deg.C, 29 deg.C or 30 deg.C, such as 0.1mbar, 0.11mbar, 0.12mbar, 0.13mbar, 0.14mbar, 0.15mbar, 0.16mbar, 0.17mbar, 0.18mbar, 0.19mbar or 0.2 mbar.

The recombinant human hyaluronidase preparation is applied to preparation of drugs for assisting subcutaneous infusion.

The auxiliary subcutaneous infusion medicament at least comprises the recombinant human hyaluronidase preparation.

The subcutaneous infusion drug preferably contains a drug carrier and a drug simultaneously or separately.

The pharmaceutical carrier may be conventional in the art and preferably comprises a glucose solution, an isotonic electrolyte solution, a basic solution, a hypertonic solution, dextran, sugar syrup or a blood product. The drug carrier is considered to be a drug having a therapeutic effect in some cases of clinical use.

The agents described in the present invention may be conventional in the art and preferably include: chemotherapeutic agents, analgesics, anti-inflammatory agents, antimicrobial agents, antiamidebic agents, trichomonacides, antiparkinson agents, anti-dysentery agents, antispasmodics, antidepressants, antirheumatics, antifungal agents, antihypertensive agents, antipyretics, antiparasitics, antihistamines, alpha-adrenergic agonists, alpha blockers, anesthetics, bronchodilators, biocides, bactericides, bacteriostats, beta adrenergic blockers, calcium channel blockers, cardiovascular agents, contraceptive agents, decongestants, diuretics, sedatives, diagnostic agents, electrolyte agents, hypnotic agents, hormones, increaser agents, muscle relaxants, muscle contraceptives, ophthalmic agents, parasympathomimetics, psychostimulants, tranquilizers, sympathomimetics, urostatics, urinary tranquilizers, vaginal agents, virucides, vitamin agents, vitamins, and the like, Non-steroidal anti-inflammatory agents, angiotensin converting enzyme inhibitors, polypeptides, proteins, nucleic acids, organic molecules, hypnotics, insulin, cytokines, antibodies, and monoclonal antibodies.

2. Pharmaceutical combination

A second aspect of the invention provides a pharmaceutical combination comprising (1) an adjunctive subcutaneous infusion drug comprising a recombinant human hyaluronidase formulation as defined in the first aspect of the invention; (2) the medicine is infused subcutaneously.

The pharmaceutical composition of the present invention is preferably a kit of parts packaged as a compound preparation or a non-compound independent preparation. Preferably, a diluent or a double solvent is also included. The diluent is preferably physiological saline. The double solvent is preferably water for injection or normal saline.

2.1 auxiliary subcutaneous infusion drug

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

The volume of the recombinant human hyaluronidase liquid preparation or the volume of the lyophilized preparation after reconstitution is preferably 0.1-10.0 ml, such as 0.2ml, 0.5ml, 1ml, 1.5ml, 2ml, 2.5ml, 3.0ml, 3.5ml, 4.0ml, 4.5ml, 5.0ml, 5.5ml, 6.0ml, 7.0ml, 8.0ml, 9.0ml and 10.0 ml.

In some embodiments, the reconstituted solution of the recombinant human hyaluronidase liquid formulation or lyophilized formulation comprises 45 units to 75000 units of recombinant human hyaluronidase per 1ml of the reconstituted solution, e.g., 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, 37000 units, 38000 units, 39000 units, 40000 units, 41000 units, 43000 units, 44000 units, 42000 units, 4700 units, 450000 units, 47000 units, 46000 units, 45000 units, 46000 units, 48000 units, 49000 units, 50000 units, 55000 units, 60000 units, 65000 units, 70000 units or 75000 units of recombinant human hyaluronidase. The volume of the recombinant human hyaluronidase preparation refers to the volume of the solution of the recombinant human hyaluronidase liquid preparation or the lyophilized preparation after reconstitution, and the concentration of the recombinant human hyaluronidase preparation refers to the concentration of the solution of the recombinant human hyaluronidase liquid preparation or the lyophilized preparation after reconstitution.

2.2 subcutaneous infusion drugs

Preferably, the drug combination can also comprise subcutaneous infusion drugs; more preferably, the method for packaging the auxiliary subcutaneous infusion medicine comprises the following steps:

(1) packaging independently;

(2) a drug combination consisting of a drug carrier for subcutaneous infusion;

(3) the medicine composition is combined with subcutaneous infusion medicines.

The pharmaceutical carrier may be conventional in the art and preferably comprises a glucose solution, an isotonic electrolyte solution, a basic solution, a hypertonic solution, dextran, sugar syrup or a blood product. The drug carrier is considered as a drug having a therapeutic effect in some cases of clinical use.

The subcutaneous infusion drug 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 subcutaneous infusion drug is preferably a drug having an anti-tumor 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, av 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, Claudin-B, EGFR, EphA 30, EphB 30, FAP, FOLR 30, GD 4, Globo H, GPC 30, GPNMB, HER-1, HER-2, HER-3, MAGE-30, MUTLIN 30, MELR 30, TMWT-30, VEGF-30, TMWT-30, or VEGF-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 tumor-targeting antibody drug is an antibody drug targeting human epidermal growth factor receptor 2(Her-2), such as trastuzumab, pertuzumab, ZW25, Margetuximab, DS8201, or ZW 49.

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

Preferably, the tumor-targeting antibody drug is a CD 38-targeting antibody drug, such as daratuzumab, Isatuximab, or MOR 202.

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.

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.

The targeted antibody drug as described above may be: abagovamab, Abciximab, Actoxumab, Adalilimumab, Adecatuzumab, Afelimomab, Afurimomab, Afurimumab, Afurizumab, Anrukinumab, Apolizumab, Arciumumab, Aselizumab, Atinumab, Atilizumab, Atolizumab (Betacomb), Atorolimumab, Bapineuzumab, Basilizumab, Bavituximab, Bevituximab, Bexituzumab, Bexizumab, Octuzumab, Avitumomab, Availazumab, Avitumomab, Britumomab, Br, Gevokizumab, Girentuzumab, Glembutumazumab, Ibrucumab, Igovamab, Imiromab, Imatumab, Inclacumab, Inatuzumab ravtansine, Infliximab, Intenumgumamab, Inolimomab, Industuzumab, Inotuzumab, Lipuidumab, Lipurizumab, Lipurib, Lipurizumab, Lipurib, The drug may be any one of the drugs selected from the group consisting of Sariumab, Satumomab pendentide, Secukinumab, Seribantimab, Setoxiximab, Seviruumab, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitumab, Sonepuzumab, Sontuzumab, Stamulab, Sulesomab, Suviuzumab, Tabalumab, Tacatuzumab tetraxetan, Tadocucimab, Talizumab, Trainonemab, Taplimomab patox, Tefibuzumab, Telimonab, Telimonabumumab, Teluminazumab, Velipentazumab, Velipentalabuzumab, Tenetumumab, Tenelumab, Tenelumbia, Totuzumab, Velipurazumab, Veliptezomab, Veliumtuzumab, Veliumotizumab, Veliptezomab, Veliumotizumab, Veliumotivatuzumab, Veliumotizumab, Vetuzumab, Veliptezomab, Veliumtuzumab, or Totuzumab, Vetuzumab, or Totuzumab.

2.3 kit of parts

The kit of parts as described above in the present invention comprises the recombinant human hyaluronidase preparation as described above, and further comprises a subcutaneous infusion drug, which can be a drug carrier and a drug, either alone or in combination.

In a first embodiment, the kit contains a single specific dose of the formulation comprising 45 units/ml to 4500000 units/ml of recombinant human hyaluronidase.

A container comprising the recombinant human hyaluronidase preparation selected from a vial, a pre-filled needle; preferably a pre-filled needle.

A container comprising the recombinant human hyaluronidase formulation selected from a vial, a pre-filled needle; preferably a vial.

Preferably, the recombinant human hyaluronidase preparation comprises 150 units/ml to 500000 units/ml of recombinant human hyaluronidase.

The recombinant human hyaluronidase preparation can have a volume of 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 recombinant human hyaluronidase formulation comprises 150 units to 75000 units of recombinant human hyaluronidase per 1ml, e.g., 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, 39000 units, 40000 units, 41000 units, 42000 units, 43000 units, 44000 units, 45000 units, 46000 units, 47000 units, 48000 units, 4800 units, 60000 units, 4900 units, 55000 units, and/ml 65000 units, 70000 units or 75000 units of 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 formulation comprising between 150 units/ml and 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 preparation preferably comprises 150 units/ml to 500000 units/ml of recombinant human hyaluronidase; more preferably, it comprises 150 units/ml to 50000 units/ml of recombinant human hyaluronidase.

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 formulation comprising 150 to 4500000 units per ml of recombinant human hyaluronidase.

Wherein, the dose of the pertuzumab is preferably 600mg or 1200mg, the dose of the trastuzumab is preferably 600mg, and the recombinant human hyaluronidase preparation preferably comprises 150 units/ml-500000 units/ml recombinant human hyaluronidase; more preferably, it comprises 150 units/ml to 50000 units/ml of recombinant human hyaluronidase. Wherein, when the pertuzumab and trastuzumab are used together, the dosage of the pertuzumab is 300-1500 mg, preferably 600mg or 1200mg, and the dosage of the trastuzumab is 200-800 mg, preferably 600 mg; wherein, the concentration of the pertuzumab and/or trastuzumab can be 50-150 mg/ml; preferably, the concentration is 60-120 mg/ml;

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 formulation comprising 150 units/ml to 500000 units/ml of recombinant human hyaluronidase, and the second container contains a single fixed dose of rituximab; the dosage of the rituximab is 700-1600 mg, preferably 1400mg or 1600 mg;

wherein, the concentration of rituximab can be 50-150 mg/ml; preferably, the concentration is 60-120 mg/ml;

the recombinant human hyaluronidase preparation preferably comprises 150 units/ml to 500000 units/ml of recombinant human hyaluronidase; more preferably, it comprises 150 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 fifth embodiment, the kit comprises two single-dose containers, wherein the first container contains a single specified dose of the formulation comprising 150 units/ml to 500000 units/ml recombinant human hyaluronidase, and the second container contains a single fixed dose of trastuzumab; the dose of the trastuzumab is 200-800 mg, preferably 600 mg;

wherein, the concentration of trastuzumab can be 50-150 mg/ml; preferably 60-120 mg/ml;

the recombinant human hyaluronidase preparation preferably comprises 150 units/ml to 100000 units/ml of recombinant human hyaluronidase; more preferably, it comprises 150 units/ml to 50000 units/ml of recombinant human hyaluronidase.

In a sixth embodiment, the kit comprises two single-dose containers, wherein the first container contains a single specific dose of the formulation comprising between 150 units/ml and 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 recombinant human hyaluronidase preparation preferably comprises 150 units/ml to 100000 units/ml of recombinant human hyaluronidase; more preferably, it comprises 150 units/ml to 50000 units/ml recombinant human hyaluronidase.

In a seventh embodiment, the kit comprises two single-dose containers, wherein the first container contains a single specified dose of said formulation comprising 150 units/ml to 500000 units/ml recombinant human hyaluronidase, and the second container contains a single fixed dose of daratumab and/or MOR 202.

Wherein, the concentration of the darunavir and/or MOR202 can be 60-120 mg/ml.

In an eighth embodiment, the kit comprises two single-dose containers, wherein the first container contains a single specific dose of said formulation comprising between 150 units/ml and 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).

In a ninth embodiment, the kit comprises two single-dose containers, wherein the first container contains a single specified dose of the formulation comprising between 150 units/ml and 500000 units/ml recombinant human hyaluronidase and the second container contains a single fixed dose of Isatuximab.

Wherein, the concentration of Isatuximab can be 60-120 mg/ml.

3. Method for treating diseases by using kit set

A third aspect of the invention relates to a method of treating a disease using a pharmaceutical combination according to the second aspect, in particular a kit of parts thereof, administered by separate administration or by mixed administration.

In a preferred embodiment of the present invention, the step of administering said combination to a subject is:

the required solution to be infused is prepared by adding or not adding required medicines into the compound preparation, and then the solution to be infused is administered to the treated object intradermally or subcutaneously.

In another preferred embodiment of the present invention, the step of administering said combination to a subject is:

the compound preparation is dissolved in a reasonable drug carrier to prepare the required solution to be infused, and then the solution to be infused is applied to the treated object in the skin or the skin.

In another preferred embodiment of the invention, the steps in administering said kit of parts to a subject are:

(a) mixing the auxiliary subcutaneous infusion drug packaged by the independent preparation, the drug carrier packaged by the independent preparation and/or the non-rational drug to prepare the needed solution to be infused. Then administering the solution to be infused to the treated object intradermally or subcutaneously; or

(b) The drug carrier in the drug box and/or the reasonable drug are mixed to prepare the solution to be infused. The auxiliary subcutaneous infusion drug in the kit is firstly applied to the treated object in an intradermal or subcutaneous way, and then the solution to be infused is applied to the treated object.

(a) mixing the auxiliary subcutaneous infusion drug packaged by the independent preparation, the drug packaged by the independent preparation and a reasonable drug carrier to prepare a required solution to be infused, and then administering the solution to be infused into the skin or the skin of a treatment object; or

(b) The medicine in the medicine box is mixed with a reasonable medicine carrier to prepare a solution to be infused, the auxiliary subcutaneous infusion medicine in the medicine box is firstly applied to a treated object in an intradermal or subcutaneous way, and then the solution to be infused is applied to the treated object.

In another preferred embodiment of the present invention, the step of administering said adjuvant subcutaneous infusion drug to the subject is:

(a) mixing the auxiliary subcutaneous infusion medicine, a reasonable medicine carrier and/or a reasonable medicine to prepare a required solution to be infused, and then administering the solution to be infused into the skin or the skin of a treatment object; or

(b) The reasonable drug carrier and/or the reasonable drug are mixed to prepare the solution to be infused, the auxiliary subcutaneous infusion drug is firstly applied to the treated object in the skin or in the skin, and then the solution to be infused is applied to the treated object.

The separate administration specifically comprises the steps of:

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

(b) the subject is then administered the subcutaneous infusion of the drug 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 to 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 administration speed can be controlled by an infusion pump or a gravity mode;

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

the subcutaneous infusion drug 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: the recombinant human hyaluronidase preparation in the kit is mixed with a subcutaneous infusion drug and then is subcutaneously administered to the subject; the preparation contains 1000 units to 200000 units of recombinant human hyaluronidase per 1ml, such as 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, 10500 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, 170000 units, 175000 units, 180000 units, 185000 units, 190000 units, 195000 units or 200000 units of recombinant human hyaluronidase.

The subcutaneous infusion medicine for mixing is in a liquid or dry powder form.

Preferably, the enzyme activity of the recombinant human hyaluronidase 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 400mg or 1600mg rituximab injection, and subcutaneous injection is carried out after uniform mixing.

The recombinant human hyaluronidase 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, 1:100, 1: 1000.

The use of a pharmaceutical composition comprising trastuzumab, pertuzumab, and recombinant human hyaluronidase preparation of 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 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 recombinant human hyaluronidase preparation as described in any of the above for the preparation of a medicament for the treatment of a CD 20-related tumor. In a particular 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 recombinant human hyaluronidase preparation as described in any of the above for 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 a pharmaceutical composition comprising an immunoglobulin, recombinant human hyaluronidase preparation as described in any of the above in the preparation of a medicament for 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 infection.

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 a pharmaceutical composition comprising daratumab and a recombinant human hyaluronidase preparation as described in any preceding claim, for the preparation of a medicament for the treatment of 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 a pharmaceutical composition comprising a human serum albumin, recombinant human hyaluronidase preparation 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 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 recombinant human hyaluronidase preparation 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.

Preferably, the enzyme activity of the recombinant human hyaluronidase preparation is 500 units/ml, and the volume is 0.5-15 ml; when the injection is administered, the hyaluronidase preparation is firstly subcutaneously injected, and then the rituximab injection 1400mg 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 the injection within 10 minutes, improving the patient experience.

The direct administration of the liquid preparation or the redissolved solid preparation of the recombinant human hyaluronidase can promote dissipation of local edema or hematoma after operation and trauma, promote infiltration of local anesthetics, relieve pain at injection parts or accelerate absorption of hypodermic or intramuscular injection liquid medicines, relieve side reaction of hyaluronic acid injection, promote diffusion of liquid medicines, exudates or blood stored in local eyes, promote absorption of vitreous opacity, prevent bulbar adhesion after chemical conjunctival burn and eliminate related inflammatory reaction, improve reabsorption of 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 preparation subcutaneously at the scapular region, wherein the dosage of the preparation is 50-5000 units, and then the contrast agent is injected at the same position.

4.Packaging material and injection system

A fourth aspect of the invention relates to packaging materials and injection systems for stable administration of the above formulations and or kits, 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 comprises but not limited to a water needle, a single needle and a 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 polymers.

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 0.1-1 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 nanoscale needle with the height of 10-2000 mu m and the width of 10-50 mu m.

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 by subcutaneous infusion. Can improve the treatment experience of patients and avoid the side effect related to intravenous 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.

Figure 7 is a summary of the stability studies of the lyophilized formulations of recombinant human hyaluronidase in different buffers.

Figure 8 is a summary of the stability study of the lyophilized formulations of recombinant human hyaluronidase at different methionine concentrations.

Fig. 9 is a summary of the stability studies of the recombinant human hyaluronidase lyophilized formulations for different concentrations of sodium chloride, trehalose, and sucrose.

Figure 10 is a summary of the stability studies of the lyophilized formulations of recombinant human hyaluronidase at different mannitol concentrations.

Figure 11 is a summary of the stability studies of lyophilized formulations of recombinant human hyaluronidase at different surfactant concentrations.

Fig. 12 is a summary of the stability studies of the liquid formulations of recombinant human hyaluronidase at different concentrations.

FIG. 13 shows the swelling of the animals in each group after continuous infusion of physiological saline.

FIG. 14 shows the swelling of the individual groups of animals after continuous infusion of fat milk.

FIG. 15 is a photograph of the pathology of brain, heart and liver tissues after injection of saline and PH 20; wherein: (a) brain tissue of the saline control group, (b) brain tissue of the high PH20 formulation group, (c) heart tissue of the saline control group, (d) heart tissue of the high PH20 formulation group, (e) liver tissue of the saline control group, and (f) liver tissue of the high PH20 formulation group.

FIG. 16 is a photograph of the pathology of spleen, lung and kidney tissues after injection of saline and PH 20; 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. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions.

Example 1: preparation of recombinant human hyaluronidase

CHO cells are adopted to be cultured in a Dynamis serum-free culture medium in a suspension way, fed-batch controlled culture is carried out by a FeedC serum-free feed medium, and the scale of the reactor is gradually enlarged to 30L by 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 Na is supplemented₂CO₃And CO₂Controlling the pH value to be 7.0; controlling the ventilation 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 depth 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: detection and analysis of recombinant human hyaluronidase preparation

The stock solution of recombinant human hyaluronidase obtained in example 1 was changed to a liquid formulation of a different composition 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 stability and the stability data for the different liquid formulations were summarized. 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 has good stability under the condition of containing 0.01-0.1% of polysorbate 20, 0.01-0.1% of polysorbate 80 and 0.01-0.1% of poloxamer 188 as a surfactant.

As shown in fig. 5, the recombinant human hyaluronidase activity in the recombinant human hyaluronidase liquid preparation of this embodiment 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 being left at 2-8 ℃ for 12 months.

As shown in fig. 6, the more components in a protein pharmaceutical 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: study of recombinant human hyaluronidase liquid formulation

And (3) replacing the recombinant human hyaluronidase stock solution into liquid preparations with different compositions, and adjusting the concentration of the recombinant human hyaluronidase to the expected 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 include SEC purity, RP purity, enzyme activity, and the results are shown in FIGS. 1-6.

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 4: study of recombinant human hyaluronidase lyophilized preparation

Different recombinant human hyaluronidase liquid preparations are prepared firstly, then freeze-drying is carried out according to the table 1, and the stability of different freeze-dried preparations is researched after freeze-drying.

TABLE 1

As shown in FIG. 7, the buffers are 5mM and 50mM phosphate buffer, histidine buffer, acetate buffer, citrate buffer and Tris buffer, and the recombinant human hyaluronidase freeze-dried preparation provided with pH5.0, 6.0, 6.5, 7.0 and 8.0 has better stability.

As shown in FIG. 8, the lyophilized formulations of recombinant human hyaluronidase containing methionine concentrations in the range of 5, 10 and 50mM had better stability.

As shown in FIG. 9, the recombinant human hyaluronidase freeze-dried preparation containing 50-170 mM sodium chloride, 25-130 mM trehalose and 25-130 mM sucrose as protective agents has better stability.

As shown in figure 10, the recombinant human hyaluronidase freeze-dried preparation containing the protective agent mannitol with the concentration of 150-280 mM has better stability.

As shown in figure 11, the recombinant human hyaluronidase freeze-dried preparation containing the surfactant polysorbate 20 with the concentration of 0.01% -0.1%, the polysorbate 80 with the concentration of 0.01% -0.1% and poloxamer 188 with the concentration of 0.01% -0.1% has better stability.

As shown in FIG. 12, the recombinant human hyaluronidase freeze-dried preparation containing the recombinant human hyaluronidase with the concentration of 150-4500000 units/ml has good stability.

Example 5: small-sized subcutaneous infusion of large-volume liquid medicine

The experiment aims to observe the influence of subcutaneous injection of the hyaluronidase liquid preparation on subcutaneous injection of normal saline into the small pig, record the injection pressure, observe the skin swelling and deformation degree and erythema condition of an injection site, and record the time required for swelling to return to normal.

8 Bama piglets were randomly divided into 2 groups by body weight, 1-negative control group (given physiological saline), 2-recombinant human hyaluronidase group (150 units/spot), and 4 pigs per group. The infusion pressure is measured by a multi-lead physiological recorder. After administration of the hyaluronidase formulation, 50mL of the fat emulsion injection was continuously infused at a rate of 300 mL/h. During infusion, the degree of swelling and deformation of the skin and the condition of erythema at the infusion site are observed, and the time required for the swelling to return to normal is recorded.

After 150mL of physiological saline is infused into the animals of the negative control group, an obvious bulge is formed at the infusion site, the swelling score is 4.0 +/-0.0, and the time required for the bulge to return to the normal state is 125.5 +/-8.1 min. The swelling degree of the recombinant human hyaluronidase preparation after infusion is lower than that of a negative control group, the swelling score is 2.3 +/-0.5, and the swelling degree is obviously reduced (P is less than or equal to 0.001). The swelling recovery time of the recombinant human hyaluronidase preparation group is 33.3 +/-8.4 min, which is obviously lower than that of a negative control group (P is less than or equal to 0.001).

Mild to moderate erythema was seen after infusion in individual animals of the negative control group. Very mild erythema was seen after infusion in individual animals of the recombinant human hyaluronidase formulation group. No statistical difference in erythema scores was observed between the two groups (P.gtoreq.0.05).

The pressure base values before the subcutaneous infusion of the physiological saline are similar in the negative control group and the recombinant human hyaluronidase preparation group, and are respectively 1.5 +/-5.5 mmHg and 5.1 +/-3.2 mmHg, the infusion pressure of the negative control group is rapidly increased along with the increase of the infusion volume, and the infusion pressures at 25mL, 50mL, 100mL and 150mL are respectively 270.5 +/-153.1 mmHg and 235.1 +/-119.4 mmHg, 215.9 +/-95.1 mmHg and 213.7 +/-90.5 mmHg. The infusion pressure of each infusion volume of the recombinant human hyaluronidase preparation group is 171.6 +/-111.7 mmHg and 164.5 +/-107.2 mmHg, 152.2 +/-88.3 mmHg and 141.5 +/-77.7 mmHg respectively, the infusion pressures are lower than those of a negative control group, and no statistical difference exists (P is more than or equal to 0.05).

Under the experimental conditions, the small pig can reduce the subcutaneous infusion pressure after the hyaluronidase liquid preparation as the test sample is injected subcutaneously, reduce the swelling and deformation degree of the skin after infusion and the time required for recovering to be normal, and assist the rapid diffusion and absorption of the infused liquid (figure 13).

Example 6: small-sized subcutaneous infusion of large-volume liquid medicine

The experiment aims to observe the influence of subcutaneous injection of the hyaluronidase liquid preparation on subcutaneous injection of the fat emulsion injection of the small pig, record the injection pressure, observe the skin swelling and deformation degree and erythema condition of an injection site, and record the time required for swelling to recover to normal.

8 Bama piglets were randomly divided into 2 groups by body weight, 1-negative control group (given physiological saline), 2-recombinant human hyaluronidase group (150 units/spot), and 4 pigs per group. The infusion pressure is measured by a multi-lead physiological recorder. After administration of the hyaluronidase formulation, 150mL of physiological saline was continuously infused at a rate of 1200 mL/h. The degree of swelling and deformation of the skin and the condition of erythema at the infusion site were observed during infusion and the time required for the swelling to return to normal was recorded.

The result shows that after 50mL of fat emulsion injection is infused into the animals of the negative control group, an obvious bulge is formed at the infusion site, the swelling score is 4.0 +/-0.0, and the time required for the bulge to return to normal is 125.0 +/-33.9 min. The swelling degree of the recombinant human hyaluronidase preparation after infusion is lower than that of a negative control group, the swelling score is 1.8 +/-0.5, and the swelling degree is obviously reduced (P is less than or equal to 0.001). The swelling recovery time of the recombinant human hyaluronidase preparation group is 22.1 +/-6.8 min, which is obviously lower than that of a negative control group (P is less than or equal to 0.001).

The pressure base values before the subcutaneous infusion of physiological saline in the animals of the negative control group and the recombinant human hyaluronidase preparation group are similar and are respectively 3.1 +/-2.7 mmHg and 1.8 +/-2.4 mmHg, the infusion pressure of the negative control group rapidly rises along with the increase of the infusion volume, and the infusion pressures of 5mL, 10mL, 20mL, 30mL and 50mL are respectively 65.2 +/-19.6 mmHg and 84.9 +/-25.9 mmHg, 114.6 +/-27.1 mmHg, 114.8 +/-40.0 and 128.6 +/-48.2 mmHg. The infusion pressure of each infusion volume of the recombinant human hyaluronidase preparation group is 32.9 +/-13.8 mmHg, 35.7 +/-20.7 mmHg, 36.1 +/-18.1 mmHg, 35.5 +/-12.2 mmHg and 32.0 +/-11.2 mmHg respectively, the infusion pressures are lower than that of a negative control group, and no statistical difference exists (P is more than or equal to 0.05).

Under the experimental conditions, the small pig can reduce the subcutaneous infusion pressure after the hyaluronidase liquid preparation as the test sample is injected subcutaneously, reduce the swelling and deformation degree of the skin after infusion and the time required for recovering to be normal, and assist the rapid diffusion and absorption of the infused liquid (figure 14).

Example 7: recombinant human hyaluronidase liquid preparation for improving absorption speed of high-concentration protein solution for subcutaneous infusion of nude mice

The experiment aims at observing 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, recording the infusion time and calculating 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 mu L of human serum albumin is subcutaneously infused at the administration part 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 speed v (mu L/min) is calculated.

The infusion speeds of the human blood albumin solution under the water pressure of 20cm, 30cm and 40cm under the left back subcutaneous (given physiological saline) of the hyaluronidase liquid preparation group animals are 54.9 +/-6.8 mu L/min, 113.0 +/-20.1 mu L/min and 201.5 +/-48.3 mu L/min respectively; the infusion speeds of human blood albumin solution under different water pressures of the right dorsal subcutaneous side (given 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 autologous negative control group, the infusion speeds of 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 8: recombinant human hyaluronidase liquid preparation for improving absorption speed of subcutaneous rituximab solution of 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 the 3 groups, before administration, 300000 units/ml recombinant human hyaluronic acid solution is added into rituximab solution according to the proportion of 1:60, and administration is carried out after uniform mixing. After anesthesia of the animals, 2 injection points are symmetrically drawn on the back by a MARK pen, and then a hyaluronidase liquid preparation or a positive control solution is administered by subcutaneous injection on the right back, and a corresponding volume of physiological saline is administered by subcutaneous injection on the left back, as an auto-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 9: recombinant human hyaluronidase liquid preparation for improving absorption speed of nude mouse subcutaneous infusion trastuzumab solution

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 solutions under water pressure of 20cm, 30cm and 40cm under left back subcutaneous conditions (given physiological saline) in 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 10: safety evaluation 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 weight into a normal saline control group and a high concentration PH20 preparation group, each group having 5 cynomolgus monkeys per 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 general dissection and microscopic examination results of the animals of the normal saline control group and the high-concentration PH20 preparation group have no abnormal pathological changes related to the injection administration, and the histopathological pictures of the main organs are shown in figures 15-16.

During the administration period of all animals, the animals were observed 1 time daily before and after the last administration, and the injection part was observed to see if there were changes such as swelling, fever, edema, erythema, and ulceration. The results show that no abnormality is seen in the local clinical observation of all animals 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; compared with the normal saline control group, no irritation reaction related to the high-concentration PH20 preparation is observed under a microscope at the injection site of the animals in the high-concentration PH20 preparation group.

Furthermore, there was no significant abnormality in the hematological indices of all animals compared to the control group, and the hematological indices of the day following the last dose (D29) were taken as an example of WBC (. times.10) for the saline control group and the high concentration PH20 formulation group⁹/L) 9.356 + -1.615 and 9.174 + -1.569, respectively, Neut (%) 31.92 + -7.52 and 37.70 + -14.89, respectively, Lymph (%) 58.94 + -7.08 and 55.06 + -15.13, respectively, 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 ALT (U/L) of the physiological saline control group and the high concentration PH20 preparation group are respectively 33.5 +/-4.9 and 35.6 +/-5.1, the AST (U/L) of 63.4 +/-17.9 and 68.0 +/-6.3, the ALP (U/L) of 405.6 +/-81.7 and 405.6 +/-89.5, the TBil (mu mol/L) of 2.268 +/-0.516 and 2.182 +/-0.698, the LDH (U/L) of 637.4 +/-131.9 and 2 +/-111.5, the Cre (mu mol/L) of 61.6 +/-10.7 and 62.6 +/-9.5, the TG (mmol/L) of 0.080.9 and 468.9 +/-111.5, the TG (mu mol/L) of 0.6 +/-10.7 and 4830.74.68 g of 0.74 g and the Glu (Glu) of 3.74 +/-0.74 +/-3.68, respectively, and the like.

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

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Shanghai Baoji pharmaceutical Co., Ltd

<120> recombinant human hyaluronidase preparation and application thereof

<130> P20016057C

<160> 2

<170> PatentIn version 3.5

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<223> recombinant human hyaluronidase

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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

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

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

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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

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

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

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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

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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

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

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

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

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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

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

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Phe Leu Lys Pro Pro Met Glu Thr Glu Glu

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

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

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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

Claims

1. A recombinant human hyaluronidase formulation comprising: recombinant human hyaluronidase, buffer, stabilizer and non-ionic surfactant:

the concentration of the buffer is 5-50 mM;

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

the pH value of the preparation is 5.0-8.0.

2. The formulation according to claim 1, characterized in that 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;

and/or the recombinant human hyaluronidase comprises an amino acid sequence shown as SEQ ID NO.1 in the sequence table; preferably, the amino acid sequence of the recombinant human hyaluronidase is shown as SEQ ID NO.2 in the sequence table;

and/or the concentration of the stabilizer is 100-400 mM; preferably 150 to 350mM, for example, 155mM, 188mM, 193mM, 215mM, 233mM, 240mM, 263mM, 283mM, 292mM, 316mM or 330 mM.

3. The formulation of claim 1, wherein:

the concentration of the trehalose or sucrose is 10-250 mM, preferably 25-200 mM, such as 25-130 mM;

and/or the concentration of the sodium chloride is 30-250 mM, preferably 50-170 mM, such as 50mM, 90mM, 130mM, 145mM or 170 mM;

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

and/or the concentration of the mannitol is 150-300 mM, such as 165mM, 220mM or 280 mM.

4. The formulation of claim 3, wherein the stabilizer is present in a concentration of 185 to 380mM and comprises at least mannitol and methionine, and trehalose or sucrose; wherein the concentration of the mannitol is 150-280 mM, the concentration of the methionine is 5-50 mM, and the concentration of the trehalose or sucrose is 25-130 mM;

and/or the enzyme activity of the recombinant human hyaluronidase is 150 units/ml-4500000 units/ml.

5. The formulation of claim 1, wherein the buffer is one or more of histidine buffer, acetate buffer, phosphate buffer, citrate buffer, Tris buffer; for example, the buffer is a histidine buffer, an acetate buffer, a phosphate buffer, a citrate buffer, or a 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 is preferably 5-50 mM, such as 5mM, 10mM or 50 mM;

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

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

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

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

6. The formulation of any one of claims 1 to 5, wherein the stabilizer of the recombinant human hyaluronidase formulation is one of the following compositions:

(1)145mM sodium chloride and 10mM methionine;

(2)130mM sodium chloride, 53mM trehalose and 10mM methionine;

(3)130mM sodium chloride, 53mM sucrose and 10mM methionine;

(4)26mM trehalose, 280mM mannitol and 10mM methionine;

(5)30mM sodium chloride, 200mM trehalose and 10mM methionine;

(6)180mM sodium chloride, 25mM trehalose and 10mM methionine;

(7)30mM sodium chloride, 200mM sucrose and 10mM methionine;

(8)180mM sodium chloride, 25mM sucrose and 10mM methionine;

(9)130mM sodium chloride, 53mM trehalose and 5mM methionine;

(10)130mM sodium chloride, 53mM trehalose and 50mM methionine;

(11)130mM sodium chloride, 53mM trehalose and 10mM methionine;

(12)22mM mannitol, 53mM trehalose and 10mM methionine;

(13)53mM trehalose, 220mM mannitol and 10mM methionine;

(14)53mM trehalose, 220mM mannitol and 5mM methionine;

(15)53mM trehalose, 220mM mannitol and 50mM methionine;

(16)150mM mannitol, 170mM sodium chloride and 10mM methionine;

(17)220mM mannitol, 53mM trehalose and 10mM methionine;

(18)150mM mannitol, 50mM sodium chloride, 53mM trehalose and 10mM methionine;

(19)150mM mannitol, 90mM sodium chloride, 130mM trehalose and 10mM methionine;

(20)150mM mannitol, 25mM trehalose and 10mM methionine;

(21)220mM mannitol, 53mM sucrose and 10mM methionine;

(22)150mM mannitol, 50mM sodium chloride, 53mM sucrose and 10mM methionine;

(23)150mM mannitol, 90mM sodium chloride, 130mM sucrose and 10mM methionine;

(24)150mM mannitol, 25mM sucrose and 10mM methionine;

(25)132mM trehalose, 150mM mannitol and 10mM methionine.

7. The formulation of claim 6, wherein the recombinant human hyaluronidase formulation is comprised of one of the following:

(71)5000 units/ml recombinant human hyaluronidase, 10mM phosphate buffer, 26mM trehalose, 280mM mannitol, 10mM methionine and 0.02% polysorbate 20, pH 7.0;

8. Use of a formulation according to any one of claims 1 to 7 for the preparation of a medicament for assisting subcutaneous infusion.

9. The use of claim 8, wherein said adjunctive subcutaneous infusion drug comprises at least said recombinant human hyaluronidase formulation.

10. The use of claim 9, wherein the subcutaneous infusion drug comprises a drug carrier and a drug, either simultaneously or separately.

11. The use of claim 10, wherein the pharmaceutical carrier comprises a glucose solution, an isotonic electrolyte solution, a basic solution, a hypertonic solution, dextran, a sugar syrup substitute, or a blood product.

12. The use of claim 10, wherein the pharmaceutical carrier is considered a therapeutically useful drug in certain clinical situations.

13. The use of claim 10, wherein the medicament comprises: chemotherapeutic agents, analgesics, anti-inflammatory agents, antimicrobial agents, antiamidebic agents, trichomonacides, antiparkinson agents, anti-dysentery agents, antispasmodics, antidepressants, antirheumatics, antifungal agents, antihypertensive agents, antipyretics, antiparasitics, antihistamines, alpha-adrenergic agonists, alpha blockers, anesthetics, bronchodilators, biocides, bactericides, bacteriostats, beta adrenergic blockers, calcium channel blockers, cardiovascular agents, contraceptive agents, decongestants, diuretics, sedatives, diagnostic agents, electrolyte agents, hypnotic agents, hormones, increaser agents, muscle relaxants, muscle contraceptives, ophthalmic agents, parasympathomimetics, psychostimulants, tranquilizers, sympathomimetics, urostatics, urinary tranquilizers, vaginal agents, virucides, vitamin agents, vitamins, and the like, Non-steroidal anti-inflammatory agents, angiotensin converting enzyme inhibitors, polypeptides, proteins, nucleic acids, organic molecules, sleep-promoting agents, insulin, cytokines, antibodies, and monoclonal antibodies.

14. A pharmaceutical combination comprising a recombinant human hyaluronidase preparation as claimed in any one of claims 1-7 and a subcutaneous infusion drug.

15. The pharmaceutical combination of claim 14, further comprising a drug carrier for subcutaneous infusion of a drug;

preferably, the method for packaging subcutaneous infusion drugs comprises the following steps:

(1) independently packaging;

(2) a drug combination consisting of a drug carrier assisted with subcutaneous infusion;

(3) and the medicine composition consists of the medicine which is assisted with subcutaneous infusion.

16. The pharmaceutical combination according to claim 14, wherein the subcutaneous infusion drug is 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 said polypeptide is preferably insulin, a glucagon-like peptide-1 (GLP-1) analog, leuprolide, vasopressin or bacitracin;

or the subcutaneous infusion medicament is preferably a medicament with 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; the concentration of the antibody is preferably 60-120 mg/ml;

wherein, when the pertuzumab and trastuzumab are used together, the dosage of the pertuzumab is 300-1500 mg, preferably 600mg or 1200mg, and the dosage of the trastuzumab is 200-800 mg, preferably 600 mg;

when rituximab is used singly, the dose of rituximab is 700-1600 mg, preferably 1400mg or 1600 mg.

17. The pharmaceutical combination according to any one of claims 14 to 16, wherein the pharmaceutical combination is a kit of parts for a combination or non-combination of separate formulations.

18. The pharmaceutical combination according to claim 17, wherein the step of administering the combination to a subject is:

19. The pharmaceutical combination according to claim 17, wherein the step of administering the combination to a subject is:

the compound preparation is dissolved in a reasonable drug carrier to prepare the needed solution to be infused, and then the solution to be infused is applied to the skin or the skin of a treated object.

20. The pharmaceutical combination of claim 17, wherein the step of administering the kit of parts to a subject is:

(a) mixing the auxiliary subcutaneous infusion drug packaged by the independent preparation, the drug carrier packaged by the independent preparation and/or the non-rational drug to prepare the needed solution to be infused, and then administering the solution to be infused into the skin or the skin of a treatment object; or

(b) The drug carrier in the drug box and/or the reasonable drug are mixed to prepare the solution to be infused, the auxiliary subcutaneous infusion drug in the drug box is firstly applied to the treated object in an intradermal or subcutaneous way, and then the solution to be infused is applied to the treated object.

21. The pharmaceutical combination according to claim 17, wherein the step of administering the kit of parts to a subject is:

22. The pharmaceutical combination of claim 21, wherein the step of administering to the subject the adjunctive subcutaneous infusion drug is:

23. A method of treating a disease, comprising administering to a subject a recombinant human hyaluronidase formulation of any one of claims 1-7 or a pharmaceutical combination of any one of claims 14-22;

the administration is preferably separate or mixed.

24. The method of claim 23, wherein said separately administering comprises the steps of:

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

(b) subsequently administering to the subject the subcutaneous infusion of the drug in the kit;

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

25. The method of claim 24, wherein the time interval between steps (a) and (b) is less than 10 minutes, preferably 0 minutes, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes or 9 minutes, when administered subcutaneously one after the other.

26. The method of any one of claims 23 to 25, wherein the separate administrations are carried out simultaneously or sequentially by means of a triplet;

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

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

the subcutaneous infusion drug 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.

27. The method of claim 23, wherein said co-administering comprises: administering intradermally or subcutaneously to the subject after mixing the recombinant human hyaluronidase formulation in the kit with a subcutaneous infusion drug;

the subcutaneous infusion drug for mixing is preferably in liquid or dry powder form.

28. The method of any one of claims 23 to 27, 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.