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Attribution of the discrepancy between ELISA and LC-MS/MS assay results of a PEGylated scaffold protein in post-dose monkey plasma samples due to the presence of anti-drug antibodies

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Abstract

High-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) methods were developed for the quantification of a PEGylated scaffold protein drug in monkey plasma samples. The LC-MS/MS method was based on the extraction of the therapeutic protein with a water-miscible organic solvent and the subsequent trypsin digestion of the extract followed by the detection of a surrogate peptide. The assay was linear over a range of 10–3,000 ng/mL. The ELISA method utilized a therapeutic target-binding format in which the recombinant target antigen was used to capture the drug in the sample, followed by detection with an anti-PEG monoclonal antibody. The assay range was 30–2,000 ng/mL. A correlation study between the two methods was performed by measuring the drug concentrations in plasma samples from a single-dose pharmacokinetic (PK) study in cynomolgus monkeys following a 5-mg/kg subcutaneous administration (n = 4). In the early time points of the PK profile, the drug concentrations obtained by the LC-MS/MS method agreed very well with those obtained by the ELISA method. However, at later time points, the drug concentrations measured by the LC-MS/MS method were consistently higher than those measured by the ELISA method. The PK parameters calculated based on the concentration data showed that the two methods gave equivalent peak exposure (C max) at 24–48 h. However, the LC-MS/MS results exhibited about 1.53-fold higher total exposure (AUCtot) than the ELISA results. The discrepancy between the LC-MS/MS and ELISA results was investigated by conducting immunogenicity testing, anti-drug antibody (ADA) epitope mapping, and Western blot analysis of the drug concentrations coupled with Protein G separation. The results demonstrated the presence of ADA specific to the engineered antigen-binding region of the scaffold protein drug that interfered with the ability of the drug to bind to the target antigen used in the ELISA method. In the presence of the ADAs, the ELISA method measured only the active circulating drug (target-binding), while the LC-MS/MS method measured the total circulating drug. The work presented here indicates that the bioanalysis of protein drugs may be complicated owing to the presence of drug-binding endogenous components or ADAs in the post-dose (incurred) samples. The clear understanding of the behavior of different bioanalytical techniques vis-à-vis the potentially interfering components found in incurred samples is critical in selecting bioanalytical strategies for measuring protein drugs.

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Abbreviations

ADA:

Anti-drug antibody

BSA:

Bovine serum albumin

ELISA:

Enzyme-linked immunosorbent assay

HCl:

Hydrochloric acid

HRP:

Horseradish peroxidase

IgG:

Immunoglobulin G

IS:

Internal standard

LBA:

Ligand binding assay

LC-MS/MS:

High-performance liquid chromatography–tandem mass spectrometry

LLOQ:

Low limit of quantification

mAb:

Monoclonal antibody

OD:

Optical density

PBS:

Phosphate-buffered saline

PBST:

Phosphate-buffered saline containing 0.05% Tween-20

PD:

Pharmacodynamics

PEG:

Polyethylene glycol

PK:

Pharmacokinetics

QC:

Quality control

SDS:

Sodium dodecyl sulfate

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SRM:

Selective reaction monitoring

TMB:

Tetramethylbenzidine

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Author notes

  1. Shujie J. Wang

    Present address: Pfizer Inc., Eastern Point Rd, Groton, CT, 06340, USA

  2. David Wang-Iverson

    Present address: , 55 Wagner Road, Stockton, NJ, 08559, USA

  3. Russell Weiner

    Present address: Merck Research Laboratories, Rahway, NJ, 07065, USA

Authors and Affiliations

  1. Bristol-Myers Squibb, Route 206 & Province Line Road, Princeton, NJ, 08540, USA

    Steven T. Wu, Aberra Fura, Murli Krishna, Paul Morin, Guodong Chen, Timothy Olah, Adrienne Tymiak & Mohammed Jemal

  2. Bristol-Myers Squibb, 100 Beaver Street, Waltham, MA, 0245, USA

    Jochem Gokemeijer

  3. Bristol-Myers Squibb, 1 Squibb Drive, New Brunswick, NJ, 08903, USA

    Karen Price

Authors
  1. Shujie J. Wang
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  10. Timothy Olah
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Corresponding authors

Correspondence to Shujie J. Wang or Mohammed Jemal.

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Wang, S.J., Wu, S.T., Gokemeijer, J. et al. Attribution of the discrepancy between ELISA and LC-MS/MS assay results of a PEGylated scaffold protein in post-dose monkey plasma samples due to the presence of anti-drug antibodies. Anal Bioanal Chem 402, 1229–1239 (2012). https://doi.org/10.1007/s00216-011-5527-9

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  • DOI: https://doi.org/10.1007/s00216-011-5527-9

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