Here I will give a brief overview of the Erenna platform, describe how Pfizer integrated the technology in a regulated lab, rate Singulex Customer support and give my opinion on the pros and cons of the platform.
What is the Erenna®?
The Erenna® is a bench-top analytical instrument that utilizes capillary flow, laser-induced fluorescence, and a highly sensitive detection optics module to achieve single molecule counting (SMC). The instrument is just as easy to use as a microplate reader and requires little maintenance. Immunoassay reagents are supplied by Singulex in a kit format which may be customized to meet the end users requirements. Paramagnetic microparticles (MPs) are used as the solid phase for immune-capture and detection of analytes in a microplate format. Signal generated by fluorescently labeled detection molecules are counted as digital events, which corresponds to a single analyte molecule. Data is analyzed with the Erenna® software or exported for analysis with standard quantitative ELISA curve fitting software.
Application of Erenna® Technology in a Regulated Bioanalytical Lab
The need for an ultra-sensitive immunoassay to support clinical pharmacokinetic studies presented an opportunity to integrate the Erenna® in our regulated bioanalytical lab. Application of Singulex’s Erenna® technology has been primarily in the area of biomarker research in a non-regulated environment. Before the Erenna® could be used to support GLP pharmacokinetic studies, certain requirements related to Good Laboratory Practices (GLPs) and FDA guidances needed to be met. A team was formed to address these requirements. The team was comprised of a subject matter expert, staff from the regulated bioanlytical group, Singulex assay development specialists, and members of Pfizer’s business operations group. Reagent availability, consistency and storage of reagents, performance specifications, automation of liquid handling steps, throughput, sample preparation, data analysis, and assay validation were addressed.
Singulex’s custom assay development services were utilized to develop a sensitive ligand binding assay for the quantification of a therapeutic protein in human plasma. Screening experiments for selection of the optimal capture and detection reagents were performed by Singulex. To ensure consistent lot to lot performance of the kits, Pfizer in-house reagents were selected, as commercial reagents were not consistent from lot-to-lot. Acceptance specifications including lower and upper limits of quantification (LLoQ and ULoQ), intra and inter-assay precision were met during assay validation. Stability of the bulk labeled kit reagents was also evaluated by storage at -80°C and 4°C. Assay performance was acceptable for the detector reagent stored at -80°C and all other kit reagents stored at 4°C for up to 12 months.
In anticipation of analysis of thousands of clinical study samples, throughput was a major concern. Automation of liquid handling steps and magnetic bead washes were accomplished with Agilent’s Bravo Liquid Handling platform. As with any new platform, training and vendor support were essential for successful implementation. Both Agilent field application specialists and Singulex assay development specialists worked closely with analysts to create a workflow that allowed for accurate and efficient analysis of samples. Assay throughput was increased by running samples in duplicate, instead of triplicate.
Singulex assay development specialists were responsive to assay performance feedback from Pfizer and modified kit instructions as necessary. When filtration of quality control (QC) samples prior to testing in the assay indicated poor (50%) recovery, an alternative sample preparation step was proposed. QC samples were not filtered and a minimum required dilution in clarified pooled plasma was performed for all samples to minimize matrix effects observed in individual donors. With the alternative sample preparation, QCs had acceptable recovery (80-120%). Additionally, removal of the sample filtration step helped increase throughput.
Data analysis and software validation was addressed by members of Pfizer’s business operations group. A custom tool to interface Erenna® data output was developed and validated by Pfizer. This tool helped with aspects of Part 11 compliance and integration with Watson LIMS for data analysis, reporting and storage.
A sensitive clinical assay with a lower limit of quantitation (LLOQ) of 32 pg/mL was successfully validated and is being used currently in Pfizer non-clinical and clinical programs.
Singulex Customer Support
Singulex provided extensive training on the Erenna® platform and our custom assay kit. Customer support was readily available by phone or email to answer technical questions and coordinate resupply of assay kits. Singulex was responsive to feedback and, as stated earlier, modified kit instructions to improve assay throughput and accuracy of results. When initial attempts to validate the assay were not successful, Singulex assay specialists travelled across the country (west to east coast!) to help troubleshoot the assay. A partnership between the analytical scientists from both companies formed and ultimately led to a presentation of this project at the 2011 AAPS NBC.
Pros and Cons of Erenna® Platform
In my opinion, sensitivity is the biggest pro for the Erenna®. For our application, the validated Erenna® assay was 56-fold more sensitive than a previously developed ELISA. Other pros included assay reagents prealiquoted in kit format, minimal maintenance of the Erenna® instrument, and excellent customer support from Singulex. Cons included high cost of kits, complicated workflow, low throughput, and lack of compatibility with in house automation.