May 28: Quantification of therapeutic antibodies and endogenous proteins
Use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) equipment in combination with adequate sample preparation allows for measurement of therapeutic as well as endogenous proteins with high accuracy and specificity. This was the main conclusion of Mohsin El Amrani (Department of Clinical Pharmacy) in his thesis on basis of which he received his PhD on May 28 in Utrecht.
Therapeutic monoclonal antibodies are widely used for the treatment of various diseases where conventional small molecule based drugs are not effective. For patients with inflammatory autoimmune disease (such as rheumatoid arthritis and Crohn’s disease) for example, the overproduction of TNF-α protein causes painful inflammatory symptoms. The level of TNF-α can be reduced through treatment with therapeutic monoclonal antibodies such as infliximab or adalimumab. However, some patients develop anti-drug antibodies that target these therapeutic ‘exogenous’ proteins thus reducing the drug concentration in plasma. This in turn renders the treatment ineffective. In this case quantitative proteomics is used for therapeutic drug monitoring of drug concentration in plasma. This provides the clinician useful insight to what is happening in the patient’s body and can be used to tailor the patient’s care.
Ligand binding assays
Traditionally, the quantification of endogenous or exogenous therapeutic proteins is performed by ligand binding assays such as ELISA or RIA. However the selectivity of these assay relies solely on the ability of the capturing and/or detecting antibodies to correctly bind to the target protein. Furthermore, different ligand binding assays for the quantification of a specific protein would often generate different results depending on the specificity and avidity of the antibody used in the test. Therefore, due to the lack of standardization, comparing results from different centers is difficult to perform which could affect clinical decision making. Quantitative proteomics with high resolution LC-MS/MS can solve many of the limitations found in ligand binding assay and provides additional advantages, such as enhanced sample through-put, linear dynamic range, precision, selectivity and multiplexing abilities.
Proteomics
Quantitative proteomics can also be used for endogenous proteins. Endogenous proteins are synthesized by the DNA within the cell nucleus. Endogenous proteins are the building blocks for all cells that make up the organism, they play a role in food digestion, they offer protection from pathogens and they heal the cells when damaged. The ability to determine whether these proteins are present in the required concentration in plasma can help the clinician to diagnose a disease but also to personalize the treatment plan.
This thesis provides a tutorial for quantitative proteomics with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, various examples of bioanalytical quantification of endogenous and therapeutic proteins in human plasma are presented. Novel sample purification strategies are introduced and optimized though experimental design. These methods lay down the foundation of quantitative proteomics with LC-MS/MS and can serve as templates for future method development.
PhD defense
Mohsin El Amrani (1978, Utrecht) obtained his PhD on May 28, 2020 at Utrecht University. His dissertation was titled “Quantification of therapeutic antibodies and endogenous proteins with LC-MS/MS”. Supervisors were prof. Alwin Huitema and prof. Erik Hack; co-supervisor was Erik van Maarseveen † (all division Laboratory, Pharmacy and Biomedical Genetics, UMC Utrecht).