A recombinant long-acting human hyaluronidase, its coding gene, production method and application

A technology of human hyaluronidase and hyaluronidase, applied in the field of recombinant long-acting human hyaluronidase, can solve problems such as short half-life

Active Publication Date: 2017-09-15
QINGDAO HUINUODE BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the half-life of the unmodified rHuPH20 in the blood is shorter than 1 minute, so it cannot be transfused through the blood and achieve the therapeutic effect in the body. Some people have used polyethylene glycol to chemically couple rHuPH20 to produce long-acting human hyaluronidase, and have used it Treating tumors in human clinical trials

Method used

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  • A recombinant long-acting human hyaluronidase, its coding gene, production method and application
  • A recombinant long-acting human hyaluronidase, its coding gene, production method and application
  • A recombinant long-acting human hyaluronidase, its coding gene, production method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Objective: Gene construction, expression, immunological detection, activity detection and reactor production of fusion protein PH20-HSA or PH20-IgFc.

[0040] Method: The structural gene PH20-HSA (SEQ ID No.6) or PH20-IgFc (SEQ ID No.7) was constructed into GC-rich animal cell expression vectors pMH3, 4, 5 (SEQ ID No.8) by PCR. , 9, 10). After these several expression plasmids were prepared in small quantities, they were stably transfected into CHO cells cultured in serum-free suspension. Through G418 screening, stable clones were picked manually with a pipette tip and placed in a 96-well plate. When the cell confluence is greater than 50%, replace with fresh serum-free medium; after 3-6 hours, collect medium samples, detect the expression level by anti-IgG2 Fc antibody dot hybridization or ELISA method, and select multiple clones with the highest expression level , continue to conduct serum-free culture in small conical bottom shake flasks and conduct passage stabili...

Embodiment 2

[0044] Objective: To separate and purify the fusion protein PH20-HSA or PH20-IgFc.

[0045] Method: The protein separation and purification strategy used in this example is (1) PH20-IgFc mainly adopts Protein A affinity purification; (2) PH20-HSA adopts the purification process of ion series hydrophobic blue gel.

[0046] (1) PH20-IgFc collection and purification.

[0047] After the culture medium was harvested, it was centrifuged at 5000 rpm for 6 minutes, and the centrifuged supernatant was taken for filtration. The pore sizes of the three filter membranes were 0.8 μm, 0.45 μm, and 0.22 μm, respectively. Load the sample onto the Mabselect column that has been equilibrated with equilibrium solution A (20mM Tris+100mM NaCl, pH 7.4), rinse until the baseline remains unchanged, and then use eluent B (100mM Gly+50mM Arg+0.01%Tween 80 , pH 3.5) for elution. Separation and purification of products were detected by SDS-PAGE.

[0048] (2) PH20-HSA collection and purification

[0...

Embodiment 3

[0056] Objective: Study on preparation and in vitro and in vivo activities of fusion protein PH20-IgFc or PH20-HSA

[0057] Method: The formulation of hyaluronidase fusion protein PH20-IgFc injection is as follows:

[0058] PH20-IgFc+10mM Na 2 HPO 4 +0.03%CaCl 2 +0.1%EDTA-Na 2 +145mM NaCl+0.1%HSA, pH 5.5-7.5

[0059] Dispense the 500U / ml PH20-IgFc filtered solution into 2ml ampoules, and place 25 bottles of the solution in an accelerated test incubator at 37°C with a humidity of 75±5%. Sampling respectively at 0,7,14,21,28,35,42 days according to the hyaluronidase activity detection of 2010 edition pharmacopoeia appendix, stability test result (table 1, figure 1 ) showed that from the 28th day, the activity of hyaluronidase decreased significantly (about 15%).

[0060] Table 1 Results of PH20-IgFc stability test at 37°C

[0061] time (d)

1

2

3

Average (U / ml)

deviation

cv

0

468

512

491

490.33

22.01

4.49

7

54...

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Abstract

The invention discloses a recombinant long-acting human hyaluronidase. The recombinant long-acting human hyaluronidase is a fusion protein PH20-HSA or PH20-IgFc of a human hyaluronidase PH20 extracellular part and a human protein (albumin HSA fragment or immunoglobulin IgG2Fc fragment), and the amino acid sequences of the PH20-HSA and the PH20-IgFc are represented by SEQ ID No. 4 and SEQ ID No. 5 respectively, and have the advantages of high in-vivo safety and long half-life. The invention also discloses an encoding gene, an expression vector, a host cell, a production method and a stable preparation of the recombinant long-acting human hyaluronidase. The achievement of industrial level and the obtaining of purified products with active energy are realized through expressing by using GC-rich high expression system and through producing Chinese hamster cells (CHO). The invention further discloses an application of the recombinant long-acting human hyaluronidase in cooperative dosage and the production of micro-molecular hyaluronic acid.

Description

technical field [0001] The invention mainly relates to the field of biotechnology, in particular to a recombinant long-acting human hyaluronidase, its coding gene, production method and application. Background technique [0002] Hyaluronic acid is the main mucopolysaccharide form in the dermis. It is a linear polysaccharide composed of repeating disaccharide units D-glucuronic acid and N-acetylglucosamine. Hyaluronic acid can retain more than 500 times its own weight in water, forming a very viscous solution, filling the collagen fiber skeleton of the extracellular matrix, and affecting the mass transfer characteristics of the extracellular matrix. The extracellular matrix is ​​a major obstacle to the subcutaneous delivery of many drugs. A solid matrix of collagen fibers embedded with a viscous gel rich in hyaluronic acid creates a certain resistance to mass transfer, limiting pharmacokinetics and the volume of fluid that can be injected subcutaneously from the same site an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A61K38/47A61K47/68A61K47/64
CPCA61K38/00C07K2319/30C07K2319/31C12N9/2474C12P19/26C12Y302/01035
Inventor 惠觅宙刘珊吴书音谢波张欣
Owner QINGDAO HUINUODE BIOLOGICAL TECH CO LTD
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