Supplementary Materials for:
Pharmacokinetic and Pharmacodynamic Evaluation of Elosulfase Alfa, an Enzyme
Replacement Therapy in Patients With Morquio A Syndrome
Clinical Pharmacokinetics
Yulan Qi, Donald G. Musson, Becky Schweighardt, Troy Tompkins, Lynne Jesaitis,
Adam J. Shaywitz, Ke Yang, and Charles A. O’Neill
Corresponding author:
Yulan Qi, YQi@bmrn.com
BioMarin Pharmaceutical Inc, 105 Digital Dr, Novato, CA, United States, 94949
S1. Supplementary Methods
S1.1 Pharmacokinetic (PK) Assay Method and Validation
The elosulfase alpha PK assay is a validated quantitative electrochemiluminescence
(ECL) sandwich assay that uses a Meso Scale Discovery (MSD) plate coated with rabbit
anti-recombinant human N-acetylgalactosamine-6-sulfatase (anti-rhGALNS) polyclonal
antibody for capture of elosulfase alpha in human plasma and goat anti-rhGALNS
polyclonal antibody labeled with a ruthenium metal chelate (MSD SULFO-TAG) for
detection. The ruthenium label produces a signal when electrochemically stimulated that
is proportional to the level of elosulfase alpha present in a sample. Plasma concentrations
were interpolated from a calibration curve generated in pooled plasma and analyzed using
a 4-parameter logistic regression model with 1/Y weighting. The intra- and inter-assay
accuracy (absolute value of percent relative error or %RE) and precision (percent
coefficient of variation or %CV) of the lower limit of quantitation (LLOQ, 15.6 ng/mL)
and upper limit of quantitation (ULOQ, 1000 ng/mL) positive controls during validation
defined the extent of the quantitative range. Inter-assay performance of the calibrator
curve from 15.6 to 1000 ng/mL was confirmed by individual calibrator RE and CV
values of 7.7% or below and a mean R-squared value of 0.9985 (0.11% CV). Incurred
sample reproducibility was demonstrated by reanalysis of approximately 10% of MOR004 study samples. The relative differences of 100 out of 112 samples reanalyzed
(89.3%) were ≤ 30.0% of the original and reanalysis mean concentration.
S1.2 Urine Keratan Sulfate Assay Method and Validation
Keratan sulfate (KS) in urine samples was enzymatically digested by keratanase II prior
to tandem mass spectrometry analysis, producing 2 major disaccharides, Gal14GlcNAc(6S) and Gal(6S)1-4GlcNAc(6S). After digestion, internal standards, heavy
isotope-labeled Gal1-4GlcNAc(6S) and heavy isotope-labeled Gal1-4GlcNAc(6S),
were added to the samples and large molecules (eg protein) were removed by
centrifugation through 5,000 or 10,000 nominal molecular weight limit spin filters.
Processed samples were chromatographed on a Hypercarb column (50 × 2.1 mm, 5 μm,
Thermo) with gradient elution and the eluant transferred directly to a triple quadrupole
mass spectrometer (API 4000) operating in negative electrospray ionization mode. The
peak of each disaccharide and internal standard was integrated and disaccharide-tointernal standard peak area ratio was used for quantification. The concentrations of
disaccharides in unknown samples were interpolated from an unweighted power curve of
standards of known concentration. The validated inter-day accuracy (% nominal
concentration) of the disaccharide calibrators from the LLOQ to the ULOQ
concentrations (0.104-20 μg/mL for each disaccharide) ranged from 94% to 106% and
the signal to noise ratios of the disaccharide LLOQ calibrators were greater than 5.
Disaccharide positive control intra- and inter-day precision ranged from 1% to 5%
relative standard deviation (RSDa). The intra- and inter-day precision of KS enzyme
digestion ranged from 1% to 6% RSD. The method was developed, validated, and
performed by Bay Bioanalytical Laboratories, Inc.
a
RSD and %CV are both calculated using the equation: 100 x (standard deviation/mean)
S1.3 Total anti-elosulfase antibody (TAb) assay method and validation
The presence of total antibodies to elosulfase alpha in human serum was determined
using a validated qualitative ECL bridging immunoassay. Serum antibodies that bind to
and bridge biotin-conjugated rhGALNS and SULFO-TAG-labeled rhGALNS were
captured via the biotin label on a streptavidin-coated MSD plate and detected by the
SULFO-TAG label upon electrochemical stimulation. TAb-positive samples were
confirmed for elosulfase alfa specificity by competition with excess unlabeled drug and
serially diluted to obtain a titer. Because a human elosulfase alpha–specific antibody was
not available, a goat anti-rhGALNS polyclonal antibody was used as a surrogate positive
control in pooled human serum. Assay sensitivity was determined to be 5.6 ng/mL of
positive control antibody and validation intra- and inter-assay precision of the positive
controls was 11.2% CV or below. The inter-assay precision of the titer positive control
was less than ± 1 dilution of the median dilution.
S1.4 Anti-elosulfase alpha–neutralizing antibody (NAb) assay method and
validation
A validated qualitative ligand receptor-binding neutralization assay was used to
determine if patient samples with a confirmed positive TAb result could neutralize the
binding of elosulfase alpha to its receptor, the cation-independent mannose-6-phosphate
receptor (CI-M6PR), immobilized on an ELISA plate. Samples were initially preincubated with biotinylated elosulfase alpha and following incubation of the mixture on a
coated plate, receptor-bound biotinylated rhGALNS was detected with streptavidin
horseradish peroxidase and a fluorogenic peroxidase substrate, QuantaBlu. A relative
reduction in signal suggested the presence of neutralizing antibodies. Screen-positive
samples were confirmed for elosulfase alpha specificity by immunodepletion with
rhGALNS-conjugated magnetic beads. Drug-specific samples were reported as positive
and non-specific samples as negative. A goat anti-rhGALNS polyclonal antibody that
neutralizes binding to CI-M6PR was used as a surrogate positive control in pooled human
serum. The assay limit of detection was 2.16 μg/mL of positive control antibody and intra
and inter-assay precision of positive control samples during validation was 12.0 and
28.9% CV or below, respectively.