Bioanalytical platform comparison using a generic human IgG PK assay format

https://doi.org/10.1016/j.jim.2013.08.009Get rights and content

Highlights

  • We compared four LBA technology platforms for total human IgG drug measurement

  • A generic (Fc-specific) assay was developed and optimized for each platform

  • Platform comparison included assay parameters, performance and other considerations

  • MSD and Gyrolab selected as default platforms for total MAb biotherapeutics

  • Immunoaffinity LC-MS/MS explored as an alternate platform for total IgG measurement

Abstract

A comparison of four different ligand-binding assay technology platforms (ELISA, Meso Scale Discovery®, Gyros® and AlphaLISA®) was conducted using quantitative assays for the measurement of a human IgG1 monoclonal antibody (MAb) in rat serum. The assays used common reagents for Fc-specific measurement to determine total levels of a human IgG MAb drug analyte, and all were fully optimized for use on each platform. Mock MAb study samples were prepared and analyzed using all platforms to assess assay performance. Assay parameters such as sensitivity, dynamic range, minimum required dilution and sample volume as well as other considerations such as per-run cost, technology availability, requisite equipment and necessary reagent modifications were evaluated toward the determination of a default go-to assay platform for monoclonal antibody biotherapeutics in this laboratory. Based primarily on superior assay performance, Meso Scale Discovery and Gyros were selected from the four technologies evaluated as our default platforms for non-regulated (discovery) study support. As an adjunct, immunoaffinity LC-MS/MS was explored as an alternate platform for generic Fc quantitation and was found to perform similarly to the ligand-binding assays.

Introduction

With the growing number of biotherapeutics in drug development, alternative assay platforms to the traditional Enzyme-Linked Immunosorbent Assay (ELISA) have seen increased usage to meet the need for sensitive and accurate measurement of drug products. Technologies such as Meso Scale Discovery® (MSD), Gyros®, AlphaLISA® and LC-MS/MS have all been put forth as viable options for the quantitation of macromolecules in various matrices. Each technology platform claims an array of advantages over the ELISA, including increased sensitivity and dynamic range, lower sample volume and/or decreased matrix interference. These factors, along with others described herein, play an important role in the selection of an appropriate platform for quantitative (Pharmacokinetic, PK) assay development and use.

MSD technology is a micro-titer, plate-based format utilizing an electrochemiluminescent (ECL) readout. Potential advantages of the MSD include decreased matrix effects, increased dynamic range and increased sensitivity.

The Gyrolab™ platform is a semi-automated system that uses microfluidics technology, centrifugal force and rotation to create a miniaturized immunoassay. The immunoassay is built on streptavidin-bead affinity columns within chambered CDs. A short assay time, automated back-end, small sample and reagent volume and increased sensitivity are a few of the possible benefits of Gyros technology.

The AlphaLISA is a homogeneous, proximity assay format employing donor and acceptor beads and a luminescent readout. The homogeneous nature of the AlphaLISA platform affords the bioanalyst with a short assay time and no wash steps.

Immunoaffinity (IA)-LC-MS/MS approaches for drug measurement provide alternative bioanalytical options when immunoassays are not available or lack sufficient specificity or sensitivity. IA-LC-MS/MS methods typically quantify one or more specific (proteotypic) peptide sequences of the drug generated enzymatically as part of the assay procedure. As in ligand-binding assays (LBAs), immunoaffinity capture can be performed using a broad array of reagents such as antibodies, receptors or ligands. One major advantage of LC-MS/MS is its high specificity and the potential for simultaneous identification and quantitation of peptides from different regions of the drug.

Other bioanalytical laboratories have compared some of the aforementioned technologies in previous publications. In one example using MSD technology (Kuhle et al., 2010), Kuhle et al developed a quantitative ECL assay for a neurofilament heavy chain protein with improved assay performance and a greater than eighty-fold increase in sensitivity over the ELISA. A luminescent AlphaLISA for the measurement of insulin has been developed with fifty-fold greater sensitivity and five-fold smaller sample volume requirements versus an ELISA (Poulsen and Jensen, 2007). A Gyros assay measuring rituximab showed sensitivity comparable to an ELISA but a ten-fold wider dynamic range (Liu et al., 2012). A comparison between ELISA and LC-MS/MS methods, both developed to measure a PEGylated scaffold protein, showed comparable assay sensitivity and dynamic range (Wang et al., 2012). Another group compared assays over several different platforms, including ELISA and MSD, with varying sensitivity and dynamic range (Soderstrom et al., 2011).

In order to aid in the selection of a go-to or default LBA platform for PK assays, an evaluation of different technologies (ELISA, MSD, Gyros and AlphaLISA) was conducted. For direct comparison between bioanalytical technologies, a generic (Fc-specific) quantitative assay for the measurement of total levels of a control human IgG1 monoclonal antibody (MAb) in Sprague–Dawley rat serum was developed on each platform. Each assay utilized the same capture and detector reagents, modified as appropriate for the respective technologies, and each was fully optimized for the specific platform. Following development, each method was qualified according to our laboratory standards for non-regulated PK LBAs. LBA qualification standards included assessment of accuracy and precision, selectivity, specificity and matrix interference. After LBA qualification, mock samples, prepared with the control human IgG at various concentrations, were analyzed in each assay to simulate study samples. The results were then assessed across all platforms for consistency and accuracy. In addition, an IA-LC-MS/MS approach was explored for generic Fc quantitation and used to analyze the mock samples.

Assay parameters used for comparison of the technology platforms included assay sensitivity and dynamic range, required sample volume, actual assay time and mock sample analysis results. Other factors considered were per-run assay cost, technology availability, special equipment required and reagent modification needs. Our goal was to assess the pros and cons for each LBA platform and use the findings from this comparison to guide in the selection of a go-to platform(s) for the development of quantitative assays for the measurement of MAb biotherapeutics.

Section snippets

Reagents, assay materials and serum

Control human IgG1 was supplied by Pfizer, Inc. Pooled and individual Sprague–Dawley rat sera were purchased from Bioreclamation (Westbury, NY). Mouse anti-human IgG Fc JDC-10 clone and biotinylated mouse anti-human IgG Fc JDC-10 clone were purchased from Southern Biotech (Birmingham, AL). Costar High-Binding 96-well flat-bottom microtiter plates (3590 plates) were purchased from Corning, Inc. (Rochester, NY). 3,3′,5,5′-tetramethylbenzidine (TMB) was purchased from BioFX Laboratories (Owings

Results and discussion

The various assay formats and platforms assessed are summarized in Fig. 1. Quantitation of the human IgG control MAb used Fc-specific reagents, and measurements represented the total amount of drug present in the samples.

Conclusion

The factors involved in the selection of an appropriate immunoassay platform for quantitative measurement of biotherapeutics may be myriad and most likely vary for each bioanalytical laboratory. Here, we have presented an evaluation of several different LBA platforms available to the bioanalyst, and after taking into consideration some of the more common parameters of relevance, have identified MSD and Gyros technology as our default go-to, ligand-binding assay platforms for MAb

Acknowledgments

We would like to thank Terri Caiazzo, Rebecca Crisino, Dawn Dufield, Rob Durham, Boris Gorovits, Sheldon Leung, Tom Miller, Franklin Spriggs and Wendy VanScyoc for their advice and assistance.

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