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Recombined human hyaluronidase, production and purification method and preparations thereof, use method and application

A technology of human hyaluronidase and human hyaluronidase, which is applied in the field of recombinant human hyaluronidase and can solve problems such as difficult high expression and production

Inactive Publication Date: 2013-12-25
惠觅宙
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recombinant human hyaluronidase PH20 has six glycosylation sites and five disulfide bonds, and it is difficult to express and produce industrially. Except for the successful expression and production of recombinant human hyaluronic acid by the American Hylozyme company using gene copy amplification technology and CHO cells Except for the enzyme PH20, there is no other report so far that has successfully expressed and produced the recombinant human hyaluronidase PH20 expressed and produced by the Hylozyme company using non-gene copy amplification technology and a new type of CHO cell (CHO-S) The case of recombinant human hyaluronidase PH20 with similar specific activity
At present, there is no use of human hyaluronidase film tablets, sprays or lotions to promote facial cosmetic nutrition, anesthetics, chemical drugs, biological drugs through the skin and mucous membranes (sublingual mucosa, buccal mucosa, soft palate mucosa, hard palate mucosa) , gingival mucosa) penetration of

Method used

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  • Recombined human hyaluronidase, production and purification method and preparations thereof, use method and application
  • Recombined human hyaluronidase, production and purification method and preparations thereof, use method and application
  • Recombined human hyaluronidase, production and purification method and preparations thereof, use method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Objective: Gene construction, expression, immunological detection, activity detection and reactor production of recombinant human hyaluronidase PH20.

[0041] Research background: Recombinant human hyaluronidase PH20 has six glycosylation sites and five disulfide bonds, which is difficult to express and produce industrially. Except for the successful expression and production of recombinant human hyaluronidase PH20 by the American Hylozyme company using gene copy amplification technology, so far there is no other report using non-gene copy amplification technology and new CHO cells (CHO-S) to successfully express and produce A case of recombinant human hyaluronidase PH20 with specific activity similar to that of recombinant human hyaluronidase PH20 expressed and produced by Hylozyme Company was produced.

[0042] The present invention utilizes our non-gene copy amplification technology with independent intellectual property rights, that is, GC-rich animal cell expressio...

Embodiment 2

[0047] Objective: To isolate and purify recombinant human hyaluronidase PH20.

[0048] Method: pre-treatment of fermentation broth: use solid-liquid separation device (centrifuge, membrane bag, hollow fiber column, sleeve, etc.) to obtain the supernatant from the fermentation broth obtained from the 5L rapid flow reactor, and tangentially in 10mM Tris pH7.5 Human recombinant hyaluronidase was purified by flow dialysis followed by Q-sepharose ion-exchange chromatography, phenyl-sepharose hydrophobic interaction chromatography, hydroxylapatite chromatography and SP-sepharose ion-exchange chromatography.

[0049] Chromatography: wash 10 column volumes with 10mM Tris+50mM NaCl pH7.5 buffer, bind human recombinant hyaluronidase to the Q Sepharose column, and then elute with the same buffer containing 400mM NaCl. The eluted peak was diluted with 2M ammonium sulfate to a final concentration of 500 mM and passed through a phenyl sepharose column. Add 10mM K 3 PO 4 to 10 mM, pH 7.4,...

Embodiment 3

[0054] Objective: To study the activity and stability of recombinant human hyaluronidase PH20

[0055] Method: The formula and stability study method of recombinant human hyaluronidase PH20 liquid preparation are as follows:

[0056]Preparation formula: 1500IU PH20+10mM Na2HPO4+0.03%CaCl2+0.1%EDTA-Na2+145mM NaCl+0.1%HSA pH5.5-7.5

[0057] Stability study: Divide the 500U / ml PH20 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%. Samples were collected on days 0, 7, 14, 21, 28, 35, and 42, and hyaluronidase activity was detected according to the appendix of the 2010 Pharmacopoeia.

[0058] result:

[0059] Table 1. Stability study of recombinant human hyaluronidase PH20

[0060] time (d)

1

2

3

Average (U / ml)

deviation

cv

0

468

512

491

490.33

22.01

4.49

7

544

420

520

494.67

65.77

13.30

1...

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Abstract

The invention discloses a recombined human hyaluronidase, a production and purification method and preparations of the recombined human hyaluronidase, a use method and application. Recombined human hyaluronidase PH20 or human hyaluronidase human albumin fusion protein PH20-HSA or human hyaluronidase human immunoglobulin IgG2Fc fragment fusion protein PH20-IgFc is adopted by the recombined human hyaluronidase and used in the mucosa or the surface of the skin. The preparations of the recombined human hyaluronidase can be made into different types such as membrane preparations, spray preparations, lotion and freeze-dried powder spray and used for skin infiltration promotion of beauty nutrient substances, skin mucosa infiltration promotion of surface anesthetic, infiltration promotion of skin disease therapeutic medicine, mucosa infiltration promotion of biological tranquillizer, mucosa skin infiltration promotion of growth factors, mucosa infiltration promotion of hypoglycemic drug, mucosa nasal cavity infiltration promotion of nervous centralis nutrient substances and the like.

Description

technical field [0001] The invention relates to a recombinant human hyaluronidase, its production and purification method, preparation, use 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 and reach blood vessels. speed and q...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42A61K47/30
Inventor 惠觅宙
Owner 惠觅宙
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