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Comparison of Neutralizing Antibody Assays for Receptor Binding and Enzyme Activity of the Enzyme Replacement Therapeutic Naglazyme® (Galsulfase)

The AAPS Journal volume 10, pages 439–449 (2008)Cite this article

Abstract

Most patients receiving Naglazyme® (galsulfase, rhASB) enzyme replacement therapy for mucopolysaccharidosis type VI develop an antibody response. To evaluate the impact of this response, two in vitro neutralizing antibody (NAb) assays were developed based on the two steps of the mechanism of action. Neutralization of enzyme activity was detected by inhibition of rhASB cleavage of a fluorogenic substrate. Neutralization of receptor binding was detected by decreased binding of labeled rhASB to immobilized soluble receptor. For the enzyme activity NAb assay, serum pretreatment was required to isolate antibodies from interfering phosphate ions, with sensitivity of ≤5 μg/mL. The receptor binding NAb assay used a five-fold dilution, with sensitivity of ≤40 μg/mL. Cutpoints for percent inhibition were based on 95% confidence intervals from naïve 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 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. In vitro NAb assays based on a well-understood mechanism of action provide specific information about the NAb mechanism.

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Abbreviations

4-MU:

4-methylumbelliferone

4-MUS:

4-methylumbelliferyl sulfate

BSA:

bovine serum albumin

CIMPR:

calcium-independent mannose-6-phosphate receptor

DPBS:

Dulbecco’s phosphate buffered solution

FDA:

Food and Drug Administration

MPS VI:

mucopolysaccharidosis type VI, Maroteaux–Lamy syndrome

NAb:

neutralizing antibody

rhASB:

recombinant human Arylsulfatase B, recombinant human N-acetyl-galactosamine 4-sulfatase, Naglazyme

RT:

room temperature

SA-HRP:

streptavidin-horseradish peroxidase

sCIMPR:

soluble domain of CIMPR

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Acknowledgements

The authors would like to thank Dawn Devereaux, Rebecca Dunham, Virginia Kalagorgevich, and Jessie Lam for additional testing support. Dr. Gary Taniguchi provided critical information about prior unsuccessful attempts at assay validation. The support from Computer Systems Validation, Instrument Validation, Analytical Chemistry, and Documentation Control at BioMarin Pharmaceutical Inc. was critical to the successful completion of this work.

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Affiliations

  1. BioAnalytical Sciences, BioMarin Pharmaceutical Inc., 105 Digital Drive, Novato, California, 94949, USA

    Joleen T. White, Lisa Argento Martell, Ryan Boyer, Lucy Crockett, Christopher Cox, Andrea Van Tuyl & Erik Foehr

  2. Analytical Chemistry, BioMarin Pharmaceutical Inc., Novato, USA

    William S. Prince & Allora Aguilera

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  1. Joleen T. White
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  2. Lisa Argento Martell
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Correspondence to Erik Foehr.

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Joleen T. White and Lisa A. Martell contributed equally to this work.

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White, J.T., Argento Martell, L., Prince, W.S. et al. Comparison of Neutralizing Antibody Assays for Receptor Binding and Enzyme Activity of the Enzyme Replacement Therapeutic Naglazyme® (Galsulfase). AAPS J 10, 439–449 (2008). https://doi.org/10.1208/s12248-008-9048-1

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Key words

  • enzyme replacement therapy
  • immunogenicity
  • naglazyme
  • neutralizing antibody
  • MPS VI