MicroLC-HRMS selective quantification of a biotherapeutic fusion protein in plasma and cerebrospinal fluid of patients with Lafora disease using a linker-containing signature peptide.

Although mass spectrometry-based approaches are increasingly used for protein biotherapeutic bioanalysis, validated workflows remain limited for fusion proteins across multiple clinical matrices, including cerebrospinal fluid (CSF). We developed a microflow liquid chromatography-high-resolution mass spectrometry (HRMS) bottom-up proteomics assay for VAL-1221, an investigational fusion protein (cell-penetrating antibody Fab linked to recombinant human acid α-glucosidase). HRMS data were acquired in data-dependent acquisition (DDA) mode to support untargeted characterization of the digest and select a linker-containing signature peptide. Quantification was performed by extracted-ion chromatogram (XIC) integration of a quantifier signal, with confirmation by a qualifier signal. The method was validated in plasma and cerebrospinal fluid, including assessment of selectivity, linearity, precision and accuracy, matrix effects, recovery, and stability. The assay was linear over 68-1700 nM in plasma and 6.8-1700 nM in CSF (coefficients of determination ≥ 0.998), with lower limits of quantification of 55.8 and 5.2 nM, respectively, and precision and accuracy were within 15%. The matrix factor was 80.5% in plasma and 102.4% in cerebrospinal fluid. Applied to compassionate-use samples from five patients with Lafora disease receiving intravenous VAL-1221 (20 mg/kg every other week), VAL-1221 was detected in plasma up to 4 h post-infusion, whereas cerebrospinal fluid concentrations were below the method detection limit under the tested regimen. Overall, this workflow provides a validated, sequence-specific approach for exposure and biodistribution assessment of fusion-protein biotherapeutics in human matrices.