(M1130-01-05) Determination of the Concentration of Recombinant Human Heparan-N-Sulfatase (rhHNS) in Human Serum Using a Ligand Binding Assay (LBA)

Purpose: The recombinant human heparan-N-sulfatase (rhHNS) is a biological drug that is being developed using high-concentration protein formulation technology, designed for administration to the central nervous system. For this study, we developed a Ligand Binding Assay (LBA) method to quantitate recombinant human heparan-N-sulfatase (rhHNS) in human serum to support human clinical studies

Methods: A ligand binding assay (LBA) method was used to measure recombinant human heparan-N-sulfatase (rhHNS) concentration in human serum. The method to quantitate recombinant human heparan-N-sulfatase (rhHNS) used the following steps. A 96‑well streptavidin gold Meso Scale Discovery (MSD) plate was coated with human biotinylated monoclonal capture antibody. The plate was blocked by adding 300 μL of dilution buffer to each well and incubating for a minimum of 1 hour. Wash the plate 3 times with a wash buffer. The recombinant human heparan-N-sulfatase (rhHNS) prepared in human serum and diluted in dilution buffer 1:20. Thirty microliters of the standard, QCs, and human serum samples were added into duplicate wells on the plate and incubated for 1.5 hours with shaking. Wash the plate 3 times with a wash buffer. Sulfo-tag labelled detection antibody to each well and incubated for 1 hour with shaking. Wash the plate 3 times with a wash buffer. Add 150 uL per well of MSD read buffer T to the plate and read using MESO QuickPlex SQ120 instrument. The quantity of luminescence corrected with the level of recombinant human heparan-N-sulfatase (rhHNS) present in the human serum samples. Data acquisition was be carried out using Methodical Mind ^TM Discovery Workbench (Meso Scale Discovery, version 1.0.38 or higher), and the resulting raw data imported into Watson Laboratory Information Management System (LIMS) software (Thermo Fisher Scientific, Inc., version 7.6.1 or higher), where regression was be performed. Statistical parameters such as means, standard deviations, and coefficient of variation and relative error were exported from Watson LIMS.

Results: The LBA method for recombinant human heparan-N-sulfatase (rhHNS) showed excellent linearity using a four-parameter logistic (4PL) equation with a 1/y² weighting at concentration range from 3.08 ng/mL to 2,240 ng/mL with coefficients of determination (r²) ≥ 0.9966. For recombinant human heparan-N-sulfatase (rhHNS) LLOQ samples in human serum, the intra‑assay CV ranged from 2.6% to 4.6% and the RE ranged from -7.8% to 18.5%; the inter‑assay CV was 8.2% and the RE was 6.2%. For recombinant human heparan-N-sulfatase (rhHNS) LQC, MQC, and HQC samples in human serum, the intra‑assay CV ranged from 0.4% to 8.2% and the RE ranged from -5.1% to 11.7%; The inter‑assay CV ranged from 5.0% to 5.6% and the RE ranged from 4.0% to 6.1%. For recombinant human heparan-N-sulfatase (rhHNS) ULOQ samples in human serum, the intra‑assay CV ranged from 1.7% to 4.3% and the RE ranged from -0.9% to 4.9%; the inter‑assay CV was 3.1% and the RE was 1.8%. Selectivity demonstrated no significant interference. Dilutional linearity samples demonstrated means within 20% of nominal for all dilutions tested and no prozone effect was observed. RE for Bench-top stability samples stored at ambient temperature for 24 hours ranged from -6.1% to 5.7% for samples stored at -20°C and  -5.7% to -3.9% for samples stored at -70°C. RE for four freeze-thaw cycles ranged from -8.9% to -4.7% for samples stored at -20°C and  from -6.7% to -1.5% for samples stored at -70°C. Long term storage stability (LTSS) was established for 32 days,  RE for LTSS ranged from -4.1% to 12.3% for samples stored at -20°C and from -9.1% to 14.0% for samples stored at -70°C.

Conclusion: The LBA method for the determination of recombinant human heparan-N-sulfatase (rhHNS) in human serum has been established and fully validated for the quantitation of recombinant human heparan-N-sulfatase (rhHNS) in human serum. The method validation parameters, including selectivity, sensitivity, precision, accuracy, dilution linearity, prozone effect, and stability were evaluated. The method has been successfully used in sample analysis to support human clinical studies with good reproducibility on incurred sample reanalysis.