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D01 Two independent validations of a mutant huntingtin protein (MHTT) assay to support the clinical development of mHTT-targeting therapies in HD
  1. David Hawellek1,
  2. Stephanie Vauleon2,
  3. Katharina Schutz2,
  4. Benoit Massonnet2,
  5. Nanda Gruben3,
  6. Marianne Manchester1,
  7. Lauren Boak4,
  8. Scott Schobel4
  1. 1Biomarker and Translational Technology Group, Roche Pharma Research and Early Development (pRED), F. Hoffmann-La Roche Ltd, Basel, Switzerland
  2. 2Bioanalytical RandD, Regulated Bioanalysis, Roche Pharma Research and Early Development (pRED), F. Hoffmann-La Roche Ltd, Basel, Switzerland
  3. 3PRA Health Sciences, Early Development Services, Bioanalytical Laboratory, Assen, the Netherlands
  4. 4F. Hoffmann-La Roche Ltd, Basel, Switzerland


Background Huntington’s disease (HD) is caused by a CAG repeat expansion in the huntingtin gene, resulting in the production of toxic mutant huntingtin protein (mHTT). Quantification of mHTT in the cerebrospinal fluid (CSF) of patients with HD using a method validated as per international guidelines is critical to support the clinical development of mHTT-targeting therapies.

Aims To validate a bioanalytical method for quantifying relative mHTT levels in human CSF in two independent laboratories.

Methods The assay was optimised in a single laboratory before its transfer to a second independent laboratory. All results were generated in regulated bioanalytical environments (i.e. by Good Clinical Practice-trained personnel in Good Laboratory Practice-certified laboratories) using a bead-based sandwich ligand-binding assay with Single Molecule Counting detection on the SMCxPROTM (Merck). The ultra-sensitive assay employs the antibody pair 2B7/MW1 for capture and detection and artificial CSF as a surrogate matrix. Assay validation followed international guidelines adapted to the context of use.

Results Full assay validation performed in two independent laboratories (Roche, PRA Health Sciences) confirmed robust inter- and intra-assay accuracy and precision and a high sensitivity (lower limit of quantification =1.63 pg-eq/mL [Roche], 1.64 pg-eq/mL [PRA Health Sciences]). Parallelism and specificity experiments involving CSF from patients with HD as well as artificial CSF matrices confirmed the selectivity and specificity of the assay, and the absence of a matrix effect. Stability of mHTT in artificial CSF was found to be sufficient to accommodate bioanalysis.

Conclusions The independent method validations demonstrate that this ultra-sensitive assay can be replicated and transferred, making it a reliable and broadly relevant tool for generating biomarker data in registrational clinical trials for mHTT-lowering therapies.

Study sponsored by F. Hoffmann-La Roche Ltd.

  • huntington’s disease
  • HD
  • mHTT
  • assay
  • cerebrospinal fluid
  • CSF
  • toxic
  • mutant huntingtin protein
  • HTT-lowering

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