Compressing Drug Development Timelines with Accelerated Immunoassay

The choice of an appropriate immunoassay technology platform can mean a difference measured in days instead of months for immunoassay development time. In today's life-science environment, when time and cost efficiencies are crucial for meeting condensed development timelines, many biopharmaceutical companies are relying more on contract research organizations (CROs) with highly automated technologies to improve laboratory productivity, shorten immunoassay development and validation timelines, and speed new drugs to market.

Mark J. Cameron

The development of immunoassays for sample analysis is important to support drug safety and toxicology studies and the critical decisions that lead to advancing drug candidates. Ideally, immunoassays are developed and validated prior to initiating in-life safety assessment studies, and those assays are validated in a good laboratory practice (GLP) or a fit-for-purpose compliant manner. Placement of toxicology and drug-safety studies often hinge on the ability of the ligand-binding assay laboratory to rapidly develop and validate pharmacokinetic and/or biomarker immunoassays.

SANDWICH ELISA

The sandwich enzyme-linked immunosorbent assay (ELISA) is the traditional method used to measure analytes and requires two layers of antibodies. The antibody captures the analyte from a complex liquid matrix such as serum or plasma. The ELISA can be custom-built on a variety of platforms, maintaining its original form; that is, a capture antibody on a solid support with the analyte sandwiched between the capture and detection antibody and a reporter indicating that the sandwich has completed.

Widely available platforms for the sandwich ELISA format include the following 96-well plate colorimetric ELISAs:

• Luminex's bead-based flow cytometry platform

• Meso Scale Discovery's (MSD's) platform, based on electrochemiluminescence

• The Gyros Gyrolab, the newest, automated platform that uses a novel compact disk (CD) format.

In addition to traditional ELISA, now decades old, these three technology platforms have proliferated in the past 10 years and are commonly found at life-science companies and CROs.

COLORIMETRIC SANDWICH ELISA

The traditional ELISAs, still commonly used, are labor intensive. The assays typically have a narrow dynamic range resulting in frequent repeat analyses when the unknown sample concentration is above the limit of quantification, and are less sensitive than sandwich assays performed on other platforms. Although readily available for many analytes and species, this platform is not capable of multiplexing. The development of multiplexing formats originated from customer demand to obtain more information from smaller sample volumes. Rodent species, especially mouse, have very limited blood volumes, which places limits on the number of samples that can be run.

LUMINEX

The widely available Luminex platform is a bead-based flow cytometry system capable of multiplexing. When the assay is complete, a laser passes over the individual 5.5 µm beads. The laser excites the fluorochrome-conjugated detection antibody, and the light energy emitted is captured by a photomultiplier tube. In theory, 100 different ELISAs could be analyzed simultaneously from a single 100-µL sample.

MESO SCALE DISCOVERY

The Meso Scale Discovery (MSD) is an immunoassay platform based on electrochemiluminescence. The assays can be run in multiplex or as single sandwich ELISA. The detection antibody is labeled with a sulfo tag, a reporter that emits light when an electrical current is applied to the 96-well, carbon-based plate. The MSD platform is popular for building immunogenicity (antidrug antibody) assays. However, only MSD develops, markets, and sells ELISA kits for the platform.

GYROS

The Gyros is an automated immunoassay platform using a CD spinning disk technology, microfluidics, and a laser-based fluorescence detection system to quantitate monoclonal antibodies, drugs, proteins, and biomarkers from nanoliters of biological fluids. The immunoassay can be developed and be ready for sample analysis in three to five days, compared with other platforms that require weeks (see Case History). Instead of a well or a bead, the Gyros uses nanoliter columns packed with streptavidin beads positioned at the periphery of a plastic disk that is subsequently spun. Each column has a microfluidics channel system etched in the disk to carry reagents and buffers for washing steps. As the disk spins, centrifugal force moves the reagents at the appropriate time over the columns.

CHOOSING AN IMMUNOASSAY PLATFORM IN A TIME-CRITICAL ENVIRONMENT

When tasked with choosing the best platform and completing immunoassay development and core validation in less than two weeks, scientists must identify, purchase, and test commercial reagents for the assay. Each immunoassay that is run on the Gyros takes 70 minutes, compared with other technologies which require four hours or more, and in some cases overnight incubation while the reagents bind in each step. Scientists using Gyros can complete a number of experiments in a single day and then have a method ready in less than three days.

The sample volume for the Gyros is very low, requiring as little as six microliters, while other technologies may require as much as 100 microliters per sample. Because of the wide dynamic range of the Gyros assay, sample repeat runs are greatly reduced when compared with a traditional ELISA. Fewer sample repeat runs means greater overall efficiency.

The Luminex can run many ELISAs simultaneously (multiplex) to search for biomarkers, whereas the Gyros does not offer multiplexing capabilities. The MSD is a versatile platform commonly used for measuring antidrug-antibodies (ADAs) and MSD offers a number of multiplexing assay kits. Due to long assay run times, however, it takes two or more weeks to build a customized immunoassay on the MSD.

While commercial Gyros kits are not widely available, CROs and biopharmaceutical companies with Gyros technology custom develop assays to boost time-critical workflows, thereby meeting increasing regulatory demands on testing. For small, medium-sized, and virtual sponsor companies especially, outsourcing immunoassays to a Gyros-equipped CRO has clear advantages—fast development times, cost-savings from reduced timelines, along with no requirement for capital investment. The CRO can then perform unattended analyses at nanoliter scale, saving labor and reducing costs while conserving sample and reagents.

Mark J. Cameron is senior manager of biomarkers and immunoassay, MPI Research, 54943 North Main Street, Mattawan, MI, 49071.