CN114573715A - Recombinant long-acting human hyaluronidase as well as production method and application thereof - Google Patents

Abstract

The invention discloses a recombinant long-acting human hyaluronidase and a production method and application thereof, and relates to the technical field of hyaluronidase preparation. The recombinant long-acting human hyaluronidase is a fusion protein of a human immunoglobulin IgG1Fc fragment and the extracellular part of human hyaluronidase PH20, and the amino acid sequence of the recombinant long-acting human hyaluronidase is shown as SEQ ID No. 1. The recombinant long-acting human hyaluronidase provided by the invention has the advantages of high safety, long half-life period and high in-vivo activity, and the fused polypeptide belongs to human protein, has lower immunogenicity and is safer to use in vivo. The invention constructs a eukaryotic expression vector for expressing the PH20-IgFc fusion protein and obtains a genetic engineering CHO cell line for stably expressing the PH20-IgFc fusion protein.

Description

A kind of recombinant long-acting human hyaluronidase and its production method and application

technical field

The present invention relates to the technical field of hyaluronidase preparation, in particular to a recombinant long-acting human hyaluronidase and a production method and application thereof.

Background technique

Hyaluronic acid (HA), also known as hyaluronic acid, is a glycosaminoglycan formed by the polymerization of N-acetylglucosamine and D-glucuronic acid as disaccharide units. The glucuronic acid is connected by β-1,3 glycosidic bonds, and the disaccharide units are polymerized by β-1,4 glycosidic bonds. Due to the difference in the degree of polymerization of disaccharide units, the molecular weight distribution of hyaluronic acid is relatively broad, and natural HA mainly exists in the form of polymer hyaluronic acid (HMW HA, Mr2×10 ⁶ Da). Molecular weight is an important parameter that determines the biological function of HA. The main component of the extracellular matrix is HMW HA, and its high viscoelasticity is the source of the extracellular matrix being able to resist external compressive forces. Relying on the high viscoelasticity, HMW HA is also used in ophthalmic surgery. Low molecular weight hyaluronic acid (LMW HA, 1×10 ⁵ DaMr2×10 ⁶ Da) has good moisturizing and lubricating properties, and is widely used in cosmetics; studies have found that ultra-low molecular weight hyaluronic acid (VLMW HA, Mr1×10 ^{5 )} Da) has the effect of promoting angiogenesis, promoting wound healing, immunoregulatory activity and anti-tumor activity, and has important potential application value in the fields of body healing, immune enhancement and tumor therapy.

There are mainly three kinds of human-derived hyaluronidase: HAase 1, HAase 2 and PH20, among which PH20 is the main enzyme that degrades HA in the human body. Hyaluronidase can degrade artificially produced recombinant high molecular weight hyaluronic acid to produce low molecular weight hyaluronic acid. At present, most of the commercially available hyaluronidase is monopolized by foreign companies, and there are few types.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a recombinant long-acting human hyaluronidase and its production method and application to solve the above-mentioned technical problems.

The present invention is realized in this way:

The present invention provides a recombinant long-acting human hyaluronidase. The recombinant long-acting human hyaluronidase is a fusion protein of human immunoglobulin IgG1Fc fragment and human hyaluronidase PH20 extracellular part, and its amino acid sequence is as shown in SEQ ID No. .1 shown.

The recombinant long-acting human hyaluronidase provided by the invention has the advantages of high safety, long half-life and high in vivo activity, belongs to human protein, and is safer to use in vivo.

The present invention also provides a recombinant long-acting human hyaluronidase encoding gene, which has the sequence shown in SEQ ID No.2.

The present invention also provides an expression vector comprising the above-mentioned gene encoding the recombinant long-acting human hyaluronidase.

The present invention also provides a host cell comprising the above-mentioned gene encoding the recombinant long-acting human hyaluronidase. In an alternative embodiment, the above-mentioned host cells are selected from CHO cells.

The present invention also provides a method for producing recombinant long-acting human hyaluronidase, the method comprising: using animal cell expression vector pMH2 or pMH7 to express the gene sequence SEQ ID No.2 in Chinese hamster cell CHO to obtain the PH20- The IgFc fusion protein is obtained after separation and purification; the gene sequences of expression vectors pMH2 and pMH7 refer to the vector sequences provided by http://www.biovector.net/, which can be purchased commercially or obtained synthetically.

In a preferred embodiment of the application of the present invention, the above-mentioned PH20-IgFc is separated and purified using the Protein G affinity purification method.

The present invention also provides a recombinant long-acting human hyaluronidase preparation, which comprises the above-mentioned recombinant long-acting human hyaluronidase or the recombinant long-acting human hyaluronidase prepared by the above method, and the preparation includes 200-1200IU of recombinant Long-acting human hyaluronidase, 10-15 mM _Na2HPO4 , 0.01-0.03% _CaCl2 , 0.1-0.2% EDTA- _Na2 , 145-150 mM _NaCl , 0.1-0.2% HSA, 0.1-0.15% mannitol, And the pH value is 5-6.

For example: 200, 500, 700, 800, 900, 1000, 1200 IU of recombinant long-acting human hyaluronidase.

The invention also provides a compound preparation, including a recombinant long-acting human hyaluronidase preparation and a medicine.

The dosage form of the above compound preparation is selected from tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays and injections.

In an optional embodiment, the above compound preparation further includes a pharmaceutically acceptable salt or excipient.

In an optional embodiment, the above-mentioned drug is a solid tumor treatment drug. Solid tumors include, but are not limited to, head and neck tumors (eg, throat cancer), thoracic tumors (eg, breast cancer, lung cancer), digestive system tumors (eg, stomach cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, bile duct cancer), genitourinary system Tumors (eg ovarian cancer, cervical cancer, prostate cancer), bone tumors, central nervous system tumors, soft tissue tumors, skin and adnexal tumors, etc.

The present invention also provides the use of a recombinant long-acting human hyaluronidase or the recombinant long-acting human hyaluronidase prepared by the above method in preparing a drug for use in combination, and the drug for use in combination prepared by the human hyaluronidase is used in Its activity is delivered to human immunoglobulin-bound tissues or organs without being degraded; or it is used to deliver a specific therapeutic drug to the tissue or organ where the disease is located.

The uses of the above-mentioned combination drugs include but are not limited to: chemotherapy, radiotherapy, photosensitizers, photothermal agents or immunotherapy.

The present invention also provides the use of the recombinant long-acting human hyaluronidase or the recombinant long-acting human hyaluronidase prepared by the above method in preparing a chemical coupling agent, and the chemical coupling agent prepared by the human hyaluronidase is used for recombinant The IgFc of long-acting hyaluronidase provides a non-functional site for chemical coupling of diagnostic agents, imaging agents, drugs, therapeutic toxins, and genes into diseased tissues and organs.

In an optional embodiment, the recombinant long-acting human hyaluronidase prepared by the above method is used for subcutaneous infusion, anesthesia for periocular surgery, penetration enhancement of chemical drugs and biological drugs, and absorption of contrast agents in urography .

The present invention has the following beneficial effects:

The recombinant long-acting human hyaluronidase provided by the present invention has the advantages of high safety, long half-life and high activity in vivo. The present invention constructs a eukaryotic expression vector expressing the PH20-IgFc fusion protein, and obtains a genetically engineered CHO cell line stably expressing the PH20-IgFc fusion protein.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings that need to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

Figure 1 is a schematic diagram of the results of the stability test of the fusion protein PH20-IgFc provided by the present invention at 37°C.

Detailed ways

In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

The features and performances of the present invention will be further described in detail below in conjunction with the embodiments.

Example 1

This example provides the construction method of the gene expression vector of the fusion protein PH20-IgFc, and the expression, immunological detection, human hyaluronidase activity detection and reactor production of the fusion protein.

The construction method of gene expression vector includes:

The structural gene PH20-IgFc (SEQ ID No. 2) was constructed into expression vectors pMH2 and pMH7 by PCR method, both of which are super-capable mammalian cell expression vector plasmids based on GC-Rich mechanism, synthesized by Reagent Company get. and mini-preparation of these two expression plasmids.

The two expression plasmids prepared above were stably transfected into serum-free suspension cultured CHO cells. Stable clones were manually picked with a pipette tip into 96-well plates by G418 selection. When the cell confluence is greater than 55%, replace the serum-free fresh medium; after 3-6 hours, collect medium samples, detect the expression level by anti-IgG1 Fc antibody dot blot or ELISA method, and select multiple cells with the highest expression level. Clones were continued to be cultured without serum and passage stability studies were carried out in small conical flasks; passage-stable clones were selected and expanded in a torrent reactor.

The results showed: (1) The fusion protein PH20-IgFc was successfully expressed in CHO cells. The results of dot blot and western blot showed that the molecular weight of the band produced by the stable high expression clone of the fusion protein PH20-IgFc was correct; (2) The high expression clone Serum-free culture and passage stabilization were carried out in small pointed-bottom flasks; (3) The stable high-expression clones were expanded by fed-batch culture in a torrent reactor, and the hyaluronidase activity in each batch of harvest liquid reached 200 -1200 active units/ml, reaching the level of industrial production.

Example 2

This example provides a method for separation and purification of fusion protein PH20-IgFc. Specifically, Protein G affinity purification was used.

The above-mentioned clones with high expression of PH20-IgFc were harvested, centrifuged at 6000 rpm for 8 min, and the centrifuged supernatant was filtered. Load the sample to Native Protein G (Alkaline Phosphatase) (ab7461) that has been equilibrated with equilibration solution A (20 mM Tris+100 mM NaCl, pH 7.4), rinse until the baseline remains unchanged, and use eluent B solution (100 mM Gly +50 mM Arg + 0.01% Tween 80, pH 3.5) for elution. The separation and purification of the products were detected by SDS-PAGE.

The purity of the fusion protein PH20-IgFc is more than 95%, and the yield is more than 30%.

Example 3

This example provides a recombinant long-acting human hyaluronidase preparation of fusion protein PH20-IgFc and its in vitro and in vivo activity studies.

The recombinant long-acting human hyaluronidase preparation is an injection, and its formula is as follows:

PH20-IgFc+12mM _Na2HPO4 +0.01% _{CaCl2 +} 0.1%EDTA- _Na2 +150mM NaCl+0.2%HAS+0.1%Mannitol, pH 5-6.

Dispense the filtered solution of 600U/ml PH20-IgFc into 2ml ampoule bottles, and place 25 bottles of the solution in an accelerated test incubator with a humidity of 75±5% at 37°C. Samples were taken on the 0th, 7th, 14th, 21st, 28th, 35th, and 42nd day respectively according to the hyaluronidase activity test in the 2010 edition of the Pharmacopoeia appendix. The stability test results (Table 1, Figure 1) showed that from the 42nd day, the hyaluronic acid There was a significant decrease in acidase activity (about 12%).

Table 1PH20－IgFc 37℃ stability test results

time (days) repeat 1 repeat 2 repeat 3 Average (U/mL) deviation CV 0 582 609 573 588.00 18.73 0.03 7 610 576 615 600.33 21.22 0.04 14 593 559 585 579.00 17.78 0.03 twenty one 576 542 622 580.00 40.15 0.07 28 554 548 579 560.33 16.44 0.03 35 548 541 570 553.00 15.13 0.03 42 543 521 536 533.33 11.24 0.02

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

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Claims (10)

1. a kind of recombinant long-acting human hyaluronidase, it is characterized in that, described recombinant long-acting human hyaluronidase is the fusion protein of human immunoglobulin IgG1Fc fragment and human hyaluronidase PH20 extracellular part, its amino acid sequence As shown in SEQ ID No.1. 2. A gene encoding a recombinant long-acting human hyaluronidase, characterized in that it has the sequence shown in SEQ ID No.2. 3. An expression vector, characterized in that it comprises the gene encoding the recombinant long-acting human hyaluronidase of claim 2. 4. A host cell, characterized in that it comprises the gene encoding the recombinant long-acting human hyaluronidase of claim 2. 5. A method for producing recombinant long-acting human hyaluronidase, characterized in that the method comprises: using animal cell expression vector pMH2 or pMH7 to express gene sequence SEQ ID No.2 in Chinese hamster cell CHO to obtain PH20-IgFc fusion protein is obtained after separation and purification. 6. The method according to claim 5, wherein the PH20-IgFc is separated and purified using a Protein G affinity purification method. 7. A recombinant long-acting human hyaluronidase preparation, characterized in that it comprises the recombinant long-acting human hyaluronidase of claim 1 or the recombinant prepared by the method of any one of claims 5-6 Long-acting human hyaluronidase, the formulation comprising 200-1200 IU of recombinant long-acting human hyaluronidase, 10-15 mM _Na2HPO4 , 0.01-0.03% _CaCl2 , 0.1-0.2% EDTA _{- Na2} , 145 - 150 mM NaCl, 0.1-0.2% HSA, 0.1-0.15% mannitol, and pH 5-6. 8. a compound preparation, is characterized in that, comprises the described recombinant long-acting human hyaluronidase preparation of claim 7 and medicine; Preferably, the drug is a solid tumor treatment drug. 9. the purposes of the recombinant long-acting human hyaluronidase prepared by the method of claim 1 or the method described in any one of claims 5-6 in the preparation and use of medicine, characterized in that , and the drug prepared by the human hyaluronidase is used for delivery to the human immunoglobulin-bound tissue or organ without its activity being degraded; or for delivering a specific therapeutic drug to the tissue where the disease is located or organ. 10. Use of the recombinant long-acting human hyaluronidase of claim 1 or the recombinant long-acting human hyaluronidase prepared by the method of any one of claims 5-6 in the preparation of a chemical coupling agent, the The chemical coupling agent prepared by human hyaluronidase is used to chemically couple diagnostic agents, imaging agents, drugs, therapeutic toxins, genes into diseased tissues by providing a non-functional site through the IgFc of the recombinant long-acting hyaluronidase organ.

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