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
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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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