Most patients receiving Naglazyme? (galsulfase, rhASB) enzyme replacement therapy for mucopolysaccharidosis type VI develop an antibody response. of 40?g/mL. Cutpoints for percent inhibition were based on 95% confidence intervals from na?ve sera. Clinical samples were similarly likely to be positive in both assays than positive for neutralization of only one step in the mechanism of action. The BMS-790052 two NAb assays yielded complementary information about potential neutralization of rhASB. Relative estimated sensitivity between neutralization assays did not correlate with the number of positive clinical samples or patients. NAb assays based on a well-understood mechanism of action provide specific information about the NAb mechanism. to perform reuptake of lysosomal enzymes that have been released from cells. Antibodies that disrupt the uptake of rhASB can decrease efficacy by preventing the biopharmaceutical from reaching the site of action. If a patient has some residual enzyme, these antibodies could also inhibit reuptake and trafficking of the endogenous enzyme, which is normally scavenged by the CIMPR receptor binding. Since binding to the soluble domain of CIMPR has been demonstrated to be both necessary and sufficient for uptake and trafficking to the lysosome (15), the measurement of antibodies that inhibit receptor binding can be used as a surrogate measurement of cellular uptake and trafficking to the lysosome. Once BMS-790052 in the lysosome, rhASB catalyzes hydrolysis of the nonreducing terminal dermatan 4-sulfate ester (11,16). Removal of this sulfate allows the continued breakdown of dermatan sulfate by the other lysosomal enzymes. Antibodies that inhibit the enzymatic activity of rhASB can decrease efficacy by preventing this substrate hydrolysis. If a patient has some residual enzyme activity, these antibodies could also inhibit activity of the endogenous enzyme by trafficking to the lysosome with enzyme from reuptake by sCIMPR, possibly leading to increased pathology. To test the inhibition of rhASB by antibodies, the enzyme activity is measured in the presence or absence of patient antibodies. A fluorogenic sulfatase substrate was used rather SLRR4A than the endogenous substrate dermatan sulfate. Use of a more promiscuous small molecule substrate is feasible in the NAb assay format since no other endogenous sulfatases will be present in the assay system. Both NAb assays were adapted from analytical chemistry procedures used for either lot release or additional characterization of rhASB. The technical challenges to develop these assays illustrate some of the unique challenges for individual NAb assays. The clinical immunogenicity data illustrated that patient populations could be subdivided based on which steps of the mechanism of action were potentially neutralized. The assay development and clinical data provide further support that scientifically justified cell-free NAb assays that are based on a well-understood mechanism of action are appropriate components of a risk-based immunogenicity program. MATERIALS AND METHODS Materials Naglazyme? (rhASB) was obtained from BioMarin Pharmaceutical Inc. (Novato, CA). Individual and pooled na?ve human sera were purchased from Binding Site (San Diego, CA) and BioReclamation (Hicksville, NY). Polyclonal sheep anti-rhASB (G192) was obtained from Covance BMS-790052 (Denver, PA) and several polyclonal rabbit anti-rhASB (BP14, BP15, J8549, J8550) were obtained from Covance and Antibodies Inc. (Davis, CA). All antibodies were purified using Protein G affinity columns obtained from GE Healthcare (Piscataway, NJ) followed by affinity chromatography with an rhASB column made using a HiTrap NHS-activated HP column from GE Healthcare. Polyclonal rabbit anti-laronidase (BP13) was obtained from Covance, and was purified using Protein G affinity column. EZ-Link Sulfo-NHS-LC-Biotin was purchased from Pierce (Rockford, IL). Antibody concentrations were measured using a BCA kit and biotin quantitation was performed using an EZ Biotin Quantitation kit, both from Pierce. Immunosorp high-binding plates for the receptor binding NAb assay were acquired from Nunc (Rochester, NY). The purified soluble extracellular domain of bovine CIMPR (sCIMPR) was obtained from Dr. Peter Lobel (Piscataway, NJ) (17). Streptavidin conjugated to horseradish peroxidase (SA-HRP) was purchased from Pierce (Rockford, IL). 3,3,5,5-tetramethylbenzidine (TMB) substrate was acquired from BioRad (Hercules, CA). 4-MUS fluorogenic substrate for the enzyme BMS-790052 activity NAb assay was acquired from Sigma (St. Louis, MO). UltraLink Protein A/G resin for antibody isolation was acquired from Pierce. MultiscreenHTS HV filter plates and vacuum manifold were obtained from Millipore (Billerica, MA). Biotin Labeling of rhASB rhASB was buffer exchanged into 10?mM sodium phosphate, 150?mM NaCl, pH?7.8, and concentrated to 2?mg/mL. Biotin (2?mg/mL in water) was added at 2.5-fold molar excess challenge ratio and incubated with gentle rocking at RT for 1?h prior to quenching with 20C25% (receptor BMS-790052 binding assay was implemented (Fig.?1). A major challenge in developing the assay was determining a combination of rhASB and sCIMPR that allowed measurement of small changes in binding and was similar to the cellular uptake curves in a cell line. From early experiments for rhASB characterization, a coating concentration of 4?g/mL sCIMPR was selected, as it was the lowest coat concentration that yielded reproducible signals. Later work demonstrated that the rhASB.