Reverse-phase HPLC assay for plasminogen activators
AssigneeGenentech, Inc.
InventorYuan Xu
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Abstract
A process is described for monitoring the effectiveness of a purification process in removing plasminogen activator (PA) endogenous to Chinese hamster ovary (CHO) cells from a sample containing human tPA or variants thereof. This process comprises incubating the sample with a protease capable of specifically cleaving the Arg275-Ile276 bond of human wild-type tPA and then with denaturing/reducing agents in respective amounts effective to reduce the disulfide bonds of human wild-type tPA; subjecting the sample to a reversed-phase high-performance liquid chromatography step, and analyzing the elution profile from the chromatography step for the amount of PA endogenous to the CHO cells present therein.
Claims6
A process for quantifying the ratio of type I to type II isozymes of human wild-type tPA (SEQ ID NO:4) or a variant or variants thereof in a sample comprising same, which process comprises:
- (a) incubating the sample with a protease capable of specifically cleaving the Arg275-Ile276 bond of said human wild-type tPA;
- (b) incubating the sample from step (a) with a denaturing agent and a reducing agent in amounts effective to reduce the disulfide bonds of said human wild-type tPA;
- (c) subjecting the sample to a reversed-phase high-performance liquid chromatography step;
- and
- (d) analyzing the elution profile from the chromatography step for the relative amounts of type I and type II isozymes present.
Dependencies are inferred from the claim text.