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Development of a Cell-Based Assay for the Detection of Neutralizing Antibodies to PF-06730512 Using Homogenous Time-Resolved Fluorescence

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Abstract

The administration of biotherapeutics has the potential to induce potent immune responses. Among these responses, the production of anti-drug antibodies (ADA), including a subset of ADA referred to as neutralizing antibodies (NAb), is of heightened concern. Aside from their capacity to alter the pharmacological profile of a given biotherapeutic, NAb can also pose significant safety risks, especially in instances where an endogenous counterpart to the drug exists. As such, the inclusion of an assay to detect NAb in clinical samples is critical to the effectiveness of a tiered approach to immunogenicity assessment. PF-06730512 is a biotherapeutic protein being developed for the treatment of primary Focal Segmental Glomerulosclerosis (FSGS). To support the immunogenicity assessment of PF-06730512, a cell-based assay was developed for the detection of NAb in FSGS serum samples. Herein, we describe the development of the assay with a focus on the challenges faced, including drug and blood collection tube interferences in NAb detection. The outcome of our efforts was a robust assay capable of detecting 1 μg/mL of a NAb positive control in the presence of clinically relevant drug concentrations up to 30 μg/mL.

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Acknowledgments

We thank current and former Pfizer colleagues who supported this work: Teresa Caiazzo and Glen Miller (BMD Reagent Group) generated biotinylated PF-06730512; Minlei Zhang (BMD RegBA Group) assisted in method qualification; Wendy Siebert (Global Product Development) facilitated procurement of FSGS serum samples from the NIDDK; Allan McPhee (Clinical Assays Group) supported method implementation and validation at CRO; Dr. Chay Lim and Dr. Susan Hurst provided thoughtful clinical context; Dr. Alison Joyce provided critical review of the manuscript; Dr. Jeffrey Kopp (NIDDK) provided a subset of the FSGS serum samples.

Funding

This work was exclusively funded by Pfizer, Inc.

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Authors and Affiliations

  1. BioMedicine Design, Pfizer, Inc., 1 Burtt Road, Andover, Massachusetts, 01810, USA

    Michael Luong, Ying Wang & Boris Gorovits

  2. Centers for Therapeutic Innovation, Pfizer, Inc., Cambridge, Massachusetts, USA

    Stephen P. Berasi, Janet E. Buhlmann & Hongying Yang

Authors
  1. Michael Luong
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  2. Ying Wang
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  3. Stephen P. Berasi
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  4. Janet E. Buhlmann
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  5. Hongying Yang
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  6. Boris Gorovits
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Corresponding author

Correspondence to Michael Luong.

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Conflict of interest

This study was funded by Pfizer. All authors listed are employees of Pfizer. Part of the work in this manuscript was presented at the 2019 AAPS D360 conference as a poster.

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Luong, M., Wang, Y., Berasi, S.P. et al. Development of a Cell-Based Assay for the Detection of Neutralizing Antibodies to PF-06730512 Using Homogenous Time-Resolved Fluorescence. AAPS J 22, 56 (2020). https://doi.org/10.1208/s12248-020-0431-x

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  • DOI: https://doi.org/10.1208/s12248-020-0431-x

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