A kind of recombinant human hyaluronidase freeze-dried preparation and its preparation method and application
A technology of human hyaluronidase and freeze-dried preparations, applied in biochemical equipment and methods, freeze-dried transportation, enzymes, etc., can solve the problems of poor stability of hyaluronidase activity, inconvenient use, high cost, etc., and achieve reduction The risk of pathogen infection, the convenience of storage, and the effect of accelerating absorption
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Embodiment 1
[0044] Recombinant human hyaluronidase was obtained by CHO cell suspension culture, and gradually expanded to 30L reactor scale production through shake flask culture. CHO cells grow in the serum-free medium independently developed by our company, and are produced by fed-batch culture. The serum-free fed-batch medium independently developed by our company is selected for controlled fed-batch; when cultured to 3-4 days, the biological The amount of feed medium added to the reactor every day is 2%-5% of the actual culture volume in the bioreactor. The culture temperature was controlled at 35-37°C, and by adding 10% Na 2 CO 3 and CO 2 The pH is controlled at 7.0; the reactor ventilation is controlled at 0.015-0.15vvm; the rotational speed is controlled at 80-150rpm; the dissolved oxygen value is controlled at 20-40%. During the cell culture process, samples were taken every day to monitor temperature, pH, glucose concentration, lactic acid concentration, osmolarity and protein...
Embodiment 2
[0058] In this example, 1,800,000 IU of recombinant human hyaluronidase, as well as buffers, cryoprotectants, excipients, enzyme activity protectants, and surfactants shown in Table 4 were dissolved in water for injection, and the volume was adjusted to 6000 mL. 0.6mL / cartridges were divided into 10000 bottles, and freeze-dried according to the freeze-drying procedure shown in Table 2.
[0059] Table 4
[0060] G H I Hyaluronidase 1800000IU 1800000IU 1800000IU Tween 20 1.2g 1.2g 1.2g Mannitol 180g 240g 300g sucrose 120g 120g 120g Disodium phosphate 8.4g 8.4g 8.4g Disodium edetate 5.4g 5.4g 5.4g calcium chloride 1.8g 1.8g 1.8g
[0061] Sampling was carried out from each group of samples of G, H, and I obtained by freeze-drying, and the shape and water content of the sampling samples were determined. The results are shown in Table 5.
[0062] table 5
[0063]
[0064] Get the liquid preparation and ...
Embodiment 3
[0067] In this example, 1,800,000 IU of recombinant human hyaluronidase, as well as buffers, cryoprotectants, excipients, enzyme activity protectants, and surfactants shown in Table 6 were dissolved in water for injection, and the volume was adjusted to 6000 mL. 0.6mL / cartridges were divided into 10000 bottles, and freeze-dried according to the freeze-drying procedure shown in Table 2.
[0068] Table 6
[0069]
[0070] Sampling was carried out from each group of samples of J, K, L, M, N, and O obtained by freeze-drying, and the shape and water content of the sampling samples were determined. The results are shown in Table 7.
[0071] Table 7
[0072]
[0073] Get the liquid preparation and the sampling sample and put it into a 40°C incubator at the same time, compare the freeze-dried preparation with the liquid preparation (8.5mg sodium chloride per milliliter, 1.4mg disodium hydrogen phosphate, 1mg human serum albumin, 1.5mg L-methyl sulfide amino acid, 0.2mg Tween 8...
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