MSACL 2023 Abstract

An End-to-end and High-Throughput Workflow for Therapeutic Drug Monitoring Using Evosep MRM

Angela Mc Ardle1, Magnus Huusfeldt1, Bharath Kumar Raghuraman1,2, Dorte Bekker-Jensen1, Ole Vorm, Nicolai Bache1
1 Evosep Biosystems, Buchwaldsgade 35, 5000, Odense, Denmark2 Department of Clinical Biochemistry, Biomarker Laboratory, Odense University Hospital, J. B. Winsløws Vej 4, 5000, Odense, Denmark

Angela Mc Ardle, PhD, MSc, BSc (Presenter) Evosep Biosystems

Presenter Bio: Angie is a Senior Scientist at Evosep Biosystems where her focus is proteomics and clinical applications. She has a strong background in mass spectrometry-based proteomics and has gained experience in multi-omic biomarker studies integrating both discovery and targeted approaches. Angie is originally from Ireland and received her PhD at University College Dublin. She continued her research career at Cedars Sinai, Los Angeles before joining Evosep Biosystems based in Denmark.

Relevant Financial Disclosures (within past 24 months, reported on Jan 26, 2023) Salary Evosep Biosystems Conflict mitigation NOT REQUIRED.

INTRODUCTION: Therapeutic antibodies are increasingly used for treatment of autoimmune disease and cancer. However, it is a significant clinical problem that a substantial number of patients do not respond to these drugs e.g. 26.1 % response rate in lung cancer. Monitoring drug levels would allow for personalised dosing and account for individual metabolic clearance and early detection of drug immunogenicity and reduced efficacy. Traditionally, ELISAs have been used to monitor blood-based levels of therapeutic antibodies. However, this approach is associated with limited dynamic range and high risk of cross reactivity, especially in the context of multiplexed assays. Here we present a generic strategy and end-to-end workflow to enrich and quantify IgG based drugs using Evosep-MRM.

OBJECTIVES: To develop a fully automated high-throughput Evosep-MRM workflow for the quantification of IgG based drugs.

METHODS: 5 ul drug free plasma was spiked with infliximab (a chimeric monoclonal antibody, sold under the brand name Remicade) across a clinically relevant dynamic range (2-100 ug/ml). Additionally, samples were spiked with heavy labelled protein to support data normalisation. Magnetic beads coated with protein G were used to enrich immunoglobulins from the complex plasma matrix. Following this, proteins were denatured, reduced and alkylated using 5% SDS, 10mM TCEP and 40 mM CAA. Next, protein aggregation capture (PAC) onto magnetic microparticles was implemented, followed by on-bead trypsin digestion and loading of the resulting peptides onto Evotips. Standard curves were analysed by an MRM assay monitoring 10 peptides (heavy & light pairs) on an Agilent 6495 QqQ coupled to an Evosep One.

RESULTS: We have developed an end-to-end workflow supporting sensitive, robust and high-throughput detection of the therapeutic antibody Remicade from plasma. Using the various gradients supported by the Evosep One system (100-500 samples per day) the assay was linear from 2 to 100 ug/ml for all gradients assessed. Additionally, inter and intra-day variability was characterised in samples across 3 concentrations (high, medium, and low). In all experiments it was possible to achieve data with coefficient of variance below 20 %. Although Remicade was used to showcase the workflow we suggest an approach whereby it can be adapted and applied to support analysis of other IgG based drugs. Additionally, we explored and report different approaches to data normalisation using heavy labelled proteins. Finally, the workflow was adapted and integrated into the Opteron 2 robot. The reproducibility of the automated workflow has been characterised and benchmarked compared to the manual approach.

CONCLUSIONS: A complete and automated sample preparation workflow was developed to support high-throughput Evosep-MRM monitoring of therapeutic drug antibodies in plasma.