A Rapid Two-Step Iduronate-2-Sulfatatse Enzymatic Activity Assay for MPSII Pharmacokinetic Assessment
Clinical studies involving enzyme replacement therapies (ERTs) have increasingly utilized enzymatic activity assays to monitor efficacy and biofunction of the drug; as a result, these assays have become an important part of pharmacokinetic (PK) and pharmacodynamic assessments in ERT trials. This paper presents a two-step enzymatic activity assay for iduronate-2-sulfatase (I2S) (EC 18.104.22.168) which we have optimized to fit in 1 day and to complete in less than 6 h. The rapid assay presented here is a significant improvement over the original two-step method with run time of 24 h which spanned 2 days. The resulting 1 day assay is efficient, robust, reproducible, and better suited for use in pharmacokinetic studies. The method was fully validated in accordance with regulatory agency guidelines so that it could be implemented in PK studies. Validation of the method required additional modifications to circumvent limitations surrounding the calculation of accuracy. This challenge was overcome by developing strategies to determine both the expected and the measured values of validation samples in activity units. Subsequently, the method was validated in accordance with the FDA guidance for the validation of quantitative ligand binding assays (LBAs). Results of method development and optimization with focus on evaluations aimed at reducing the total assay run time as well as a summary of method validation performance are presented in this publication.
KeywordsEnzymatic activity assay ERT Hunter Syndrome I2S MPSII α-l-iduronidase
Mitra Azadeh, Luying Pan, and Yongchang Qiu are full time employees of Shire. Ruben Boado is a full time employee of ArmaGen. We thank Eurofins Pharma Bioanalytics Services, St Charles, MO, USA, contracted by Shire to perform valiation of the two-step activity method, for their services. We also thank the Shire Discovery Therapeutic Group for providing the IDUA.
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