Method of stabilizing alkaline phosphatase
A phosphatase, alkaline technology, applied in the field of preparations containing alkaline phosphatase, can solve the problem of freeze-dried products that do not show increased activity
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Embodiment 1
[0026] To a 20 mM POPSO-NaOH buffer solution ( pH 7.5) (1) without BSA, (2) with 1 (w / v)% BSA and (3) with 3 (w / v)% BSA, and inject 2ml of each resulting solution into a vial and freeze-dried. Under each of the above conditions, each lyophilized product was subjected to a temperature acceleration test at 37° C., and then reconstituted with 2 ml of distilled water at weekly measurements to measure ALP activity, and the test lasted up to 4 weeks. The change in the remaining activity of each sample is shown in Table 1. Significant stabilization was observed in the BSA-added system compared to the BSA-free system.
[0027] Table 1
[0028] BSA-free
Embodiment 2
[0030] 3% sucrose, 3% galactose, and 3% lactose were each added to 20 mM BES containing 3% BSA, 0.1 mM zinc chloride, 30 mM valine, and 400 U / L of human liver-derived ALP - In NaOH buffer (pH 7.5), 3 ml of each resulting solution was injected into two vials and lyophilized. Each lyophilized product was reconstituted by adding 3 ml of distilled water to measure ALP activity. The remaining one vial was placed in a 37°C incubator for 1 week. Thereafter, ALP activity was measured in the same manner. As shown in Table 2, the galactose and lactose-added products of the present invention showed higher residual activity than the sucrose-added system.
[0031] Table 2
[0032] 3% sucrose
Embodiment 3
[0034] 5 (w / v)% of galactose, 5 (w / v)% of fructose and 5 (w / v)% of lactose were added to the solution containing 3% BSA, 0.5mM magnesium chloride, 10μM zinc chloride, respectively. and 400 U / L of human liver-derived ALP (No. T-73, produced by Asahi Kasei Co, Ltd.) in 40 mM PIPES-NaOH buffer, and 2 ml of each resulting solution was injected into a vial and freeze-dried. The freeze-drying conditions are: after vacuuming at a freezing temperature of -50°C, main drying at -10°C for 12 hours, re-drying at 20°C for 24 hours, and vacuum sealing. 2 ml of distilled water was added to each lyophilized product to dissolve it to measure the ALP activity of each sweetened product. The remaining vials were placed in a 37°C incubator, and ALP activity was measured by dissolving an aliquot of the product in 2 ml of distilled water weekly for up to 3 weeks. The remaining activity is shown in Table 3, and the activity of the enzyme was 100% immediately after lyophilization. The residual activ...
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