Application of miniaturized immunoassays to discovery pharmacokinetic bioanalysis

Abstract

Introduction

Pharmacokinetic properties of biotherapeutics are an important aspect of preclinical drug development. The lead identification and optimization space is characterized by aggressive timelines, large sample numbers, a variety of species and matrices, and limited reagent and sample volumes all of which represent challenges for traditional microtiter plate assays. Since the Gyrolab immunoassay platform can accommodate small sample volumes and automated assay processing, we evaluated the workstation as an alternative to the plate-based assays.

Methods

Three representative example assays — a generic anti-human IgG, a target specific and an anti-drug capture assay — were investigated in detail for accuracy and precision performance and their application to bioanalytical support for preclinical pharmacokinetic studies. Different animal matrices were tested in the assays and during study support.

Results

Gyrolab procedures could be closely modeled after regular microtiter plate assays. The small reagent volumes necessary for Gyrolab allowed studying serial bleeds of transgenic mice with only 10 μL of blood sample. During development and during study support, the Gyrolab performance was similar to what can be expected from plate-based systems with accuracy and precision within 100 ± 20% or less.

Discussion

Overall, the technology was well suited to support quantitation of biotherapeutics using small volume samples from different preclinical species. Limited operator involvement for assay processing allowed for reduced staffing and training. However, high instrument costs and a single source of reagent supplies represent risks when moving assays further into long-term applications such as clinical studies. Despite interest in the bioanalytical field, this is the first detailed investigation of bioanalytical applications of Gyrolab in pharmacokinetic studies.

Introduction

The advent of biopharmaceuticals brought a new focus on immunoassays as a core technology for bioanalytical support of pharmacokinetic (PK) and immunogenicity studies. While immunoassays have a long history, most formats are still microtiter plate-based without technological advances comparable to small molecule analysis by liquid-chromatography–mass spectrometry (LC–MS). Plate-based immunoassays, mostly enzyme-linked immunosorbent assays (ELISA), typically require long development times of up to several weeks, large reagent and sample volumes of 100–200 μL/well, and well-trained operators to achieve high accuracy and precision (David, 2005). Complete ELISA automation using robotic liquid handlers has addressed through-put in some laboratories. However, this can be cumbersome to set up, in particular if small sample volumes need to be transferred with high accuracy, and contract research organizations often have no access to robotic equipment. As bioanalytical support is increasingly out-sourced, immunoassays should be robust and well-developed in order to minimize involvement of the sponsor in technical trouble-shooting (Ray et al., 2010).

The biopharmaceutical discovery space on the other hand is characterized by aggressive timelines, large sample numbers, a variety of animal species and sample matrices, and limited available critical reagent and sample volumes. The implementation of a flexible assay design, such as “generic” anti-human antibody assays (Stubenrauch et al., 5-1-2009, Yang et al., 2008), could address a few of these challenges. Some of the advantages of LC–MS were also attempted to be transferred into biotherapeutics development but have not quite matured yet (Ezan et al., 2009, Ezan and Bitsch, 2009). In particular, limited sample volumes still represent an obstacle for immunoassays leading to increased animal numbers per study for smaller species to accommodate volume requirements. This can become an issue when studying transgenic models with limited colony sizes. Assay miniaturization as used for biomarker discovery (Ellington et al., 2010, Jokerst et al., 2009, Templin et al., 2004) has not found wide-spread application for pharmacokinetic immunoassays.

The Gyrolab immunoassay platform (Gyros, Uppsala, Sweden) was developed to address several of the challenges outlined above. It requires minimal sample and reagent volumes, almost no hands-on time and 112 data points can be generated within 1 h. Details of the technology can be found in the manufacturer's web page (http://www.gyros.com). Briefly, immunoassays are carried out on a special compact disk (CD). Reagents and samples flow through nano-scale channels etched into the CD over a streptavidin-coated bead column where the immunosandwich is assembled. The detection antibody is fluorescently labeled to allow visualization by laser. The Gyrolab is completely integrated allowing fully automated immunoassays without operator oversight.

Although several applications of the platform have been published (Kange et al., 2005, Lund et al., 2010, Rivera et al., 2005, Eriksson et al., 2006) including immunogenicity and pharmacokinetic assays, (Singh et al., 2010, Yeung et al., 2008, Hamuro et al., 2009, Mora et al., 2010, van der Woude et al., 2010), detailed information regarding platform performance still is limited. We evaluated Gyrolab-based PK immunoassays of biotherapeutics in the discovery space with the objective of addressing limitations in sample volumes, staffing and turn-around times. In this manuscript we describe our experiences with Gyrolab performance using three representative assays.