Enzymatic activity assays for i2s
AssigneeShire Human Genetic Therapies, Inc.
InventorsPeter BERNHARDT, Claire TIEN, Elisha FIELDING, Vijay CHHAJLANI
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Abstract
In certain embodiments of the present invention, kinetic parameters of I2S enzyme are determined. In some instances, a sample including I2S enzyme is incubated under defined conditions, with a series of determined amounts of I2S substrate including a detectable label. Following incubation, the reaction mixture can be analyzed, e.g., by a method including chromatography. A detection unit can be used to measure the presence of the detectable label. Data can be analyzed to determine kinetic parameters.
Claims24
A method of determining the potency of iduronate-2-sulfatase (I2S), comprising the steps of:
- contacting a sample comprising iduronate-2-sulfatase (I2S) with a substrate containing a terminal iduronate-2-sulfate under conditions that permit the I2S-catalyzed desulfation of the substrate, wherein the I2S-catalyzed desulfation of the substrate is associated with a detectable signal;
- and
- detecting the detectable signal, thereby determining one or more kinetic parameters or the specific activity of the I2S,
- wherein the one or more kinetic parameters or the specific activity is indicative of the potency and active properties of I2S.
The method of any one of the preceding claims, wherein the substrate is 4-methylumbelliferyl-α-L-idopyranosiduronic acid 2-sulfate (IdoA2S-4MU):
The method of any one of the preceding claims, wherein the I2S-catalyzed desulfation generates an I2S product, and the product is IdoA-4MU:
The method of any one of the preceding claims, wherein the step of detecting the detectable signal comprises performing chromatography.
The method of any one of the preceding claims, wherein the step of detecting the detectable signal comprises determining the amount of the product as compared to a control.
The method of any one of the preceding claims, wherein the step of detecting the detectable signal comprises determining the rate of product formation.
The method of any one of the preceding claims, the one or more kinetic parameters are selected from the group consisting of Vmax, Km, kcat, specific activity, and combination thereof.
The method of any one of the preceding claims, wherein the sample is a drug substance, a drug product, or a stability sample of drug substance and drug product.
The method of any one of the preceding claims, wherein the conditions that permit I2S-catalyzed desulfation of the substrate comprise incubation at about 37° C. for about 20 minutes, a buffer pH of approximately 4-5, and in the presence of BSA.
The method of any one of the preceding claims, wherein the conditions that permit the I2S-catalyzed desulfation of the substrate comprise between 0 and 0.4 mg/mL BSA.
The method of any one of the preceding claims, wherein the method further comprises a step of quenching the desulfation by addition of acetonitrile, or another organic solvent, water-miscible or not miscible, or by using heat denaturation.
Dependencies are inferred from the claim text.