High throughput, fluorescence-based esterase activity assay for assessing polysorbate degradation risk during biopharmaceutical development
AssigneeGenentech, Inc.
InventorsLi YI, Adithi Chandrasekhara BHARGAVA, Inn Huam YUK
Life of this patent
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Abstract
The present disclosure provides compositions, methods, and kits for detecting lipolytic activity. In some embodiments, the composition comprises an aqueous assay sample and an organic solvent, wherein the organic solvent comprises 4-methylumbellif-erryl caprylate (MU-C8). Also provided herein are methods for determining the stability of a protein preparation.
Claims51
An assay for determining enzymatic activity of host cell proteins (HCPs) in a sample, wherein the HCPs comprises hydrolase, the assay comprising the steps of:
- a) obtaining a reaction mixture in a microplate, wherein the reaction mixture comprises:
- the sample, a reaction buffer, and a 4-methylumbelliferone carboxylate ester as a fluorogenic substrate;
- b) obtaining a negative control;
- c) exposing the reaction mixture and the negative control to fluorescence signal;
- d) monitoring conversion of the fluorogenic substrate from a non-fluorescent state to a fluorescent product in the reaction mixture resulting from exposure to the fluorescence signal, wherein the fluorescent product is 4-methylumbelliferone (MU);
- and
- e) determining and quantifying the HCP enzymatic activity based on conversion of the fluorogenic substrate in step d).
An assay for determining enzymatic activity of host cell proteins (HCPs) in a sample, wherein the HCPs comprises hydrolase, and the assay comprises the steps:
- a) obtaining a reaction mixture comprising the sample, a reaction buffer, and a fluorogenic substrate, wherein the fluorogenic substrate is 4-methylumbelliferone carboxylate ester, wherein the carboxylate ester of the 4-methylumbelliferone carboxylate ester comprises no more than ten carbons;
- b) measuring the fluorescent signal at one or more time points;
- and
- c) determining and quantifying the HCP enzymatic activity based on the measured fluorescence.
A composition comprising
- (a) an aqueous assay sample comprising a protein preparation,
- (b) an organic solvent comprising a reaction buffer, and at least one 4-methylumbelliferone carboxylate ester;
- wherein the fluorogenic substrate is 4-methylumbelliferone carboxylate ester, and wherein the carboxylate ester of the 4-methylumbelliferone carboxylate ester comprises no more than ten carbon atoms.
A method of determining stability of a protein preparation comprising
- a. obtaining a reaction mixture in a microplate, wherein the reaction mixture comprises:
- the sample, a reaction buffer, and a 4-methylumbelliferone carboxylate ester as a fluorogenic substrate;
- b. obtaining a negative control;
- c. exposing the reaction mixture and the negative control to a fluorescence signal;
- d. monitoring conversion of the fluorogenic substrate from a non-fluorescent state to a fluorescent product in the reaction mixture resulting from exposure to the fluorescence signal, wherein the fluorescent product is 4-methylumbelliferone (MU);
- and
- e. determining and quantifying the HCP enzymatic activity based on conversion of the fluorogenic substrate in step d).
A method of optimizing or selecting a protein purification process to improve removal of hydrolytic HCPs, said method comprising
- a. obtaining a reaction mixture in a microplate, wherein the reaction mixture comprises:
- the sample, a reaction buffer, and a 4-methylumbelliferone carboxylate ester as a fluorogenic substrate;
- b. obtaining a negative control;
- c. exposing the reaction mixture and the negative control to fluorescence signal;
- d. monitor conversion of the fluorogenic substrate from a non-fluorescent state to a fluorescent product in the reaction mixture resulting from exposure to the fluorescence signal, wherein the fluorescent product is 4-methylumbelliferone (MU);
- and
- e. determining and quantifying the HCP enzymatic activity based on conversion of the fluorogenic substrate in step d).
Dependencies are inferred from the claim text.