Capillary western immunoassay for sensitive and reproducible dystrophin quantification


Translational Research

Poster Number: 319


Ioannis Eugenis, PhD, Bio-Techne, Charles Haitjema, PhD, Bio-Techne, Rachel Talis, Bio-Techne, Francisco Ramirez, Bio-Techne, Kelly Gardner, PhD, Bio-Techne, Chris Heger, PhD, Bio-Techne

The quantification of dystrophin is a critical component of assessing the potency of dystrophin-restoring therapies, and the analytical methods used must be carefully validated for robust clinical trials and regulatory approval. Traditionally, dystrophin quantification has been performed by Western blot and immunofluorescence, but these methods have been inadequate in reproducibly quantifying dystrophin, which has slowed the regulatory approval of therapies for dystrophinopathies. Capillary electrophoresis coupled with immunodetection, or capillary western immunoassay, used for dystrophin quantification has demonstrated reproducibility in line with FDA and EMA regulations, with an average CV between replicate measurements below 20%, even in the lower range of dystrophin levels and between different operators. The capillary western immunoassay circumvents the gel-to-blot transfer of traditional Western blots and is an automated system, making it inherently more reproducible than Western blot and immunofluorescence methods. Moreover, the capillary immunoassay is 100X more sensitive, has a larger dynamic range, and uses less sample than a Western Blot. We further highlight the multiplex capability of the capillary western immunoassay, which enables simultaneous total protein detection to accurately normalize dystrophin quantification and eliminates the need for unreliable housekeeping controls. In addition, we demonstrate how the capillary immunoassay can be used to measure six critical quality attributes, including bioprocess contaminants, viral identity, viral titer, potency, capsid protein ratio, and empty/full content ratio, for gene therapies. Here, we propose capillary western immunoassay as the standard potency assay for dystrophin quantification to develop, manufacture, and commercialize gene therapies for dystrophinopathies.