Quanti fi cati on of therapeuti c anti LC-MS/MSproteins with bodies and endogenous c on of therapeuti ficati Quanti Amrani Mohsin El Quanti fi cati on of therapeuti c anti LC-MS/MS proteins with bodies and endogenous c on of therapeuti ficati Quanti Quanti fi cati on of therapeuti c Quanti fi cati on of therapeuti c UITNODIGINGUITNODIGING anti bodies and endogenous proteins voor het bijwoonen van anti bodies and endogenous proteins voorde hetopenbare bijwoonen verdediging van with LC-MS/MS de openbarevan het verdediging proefschrift with LC-MS/MS van het proefschrift Quanti fi cati on of Quanti fi cati on of Mohsin El Amrani therapeuti c anti bodies Mohsin El Amrani therapeutiand endogenous c anti bodies and proteinsendogenous with proteinsLC-MS/MS with LC-MS/MS op donderdag 28 mei 2020 op donderdagom 14:3028 Mei uur 2020 om 14:30 uur Academiegebouw AcademiegebouwUniversiteit Utrecht UniversiteitDomplein 29Utrecht te Utrecht Domplein 29 te Utrecht Aansluitend recepti e ter Aansluitendplaatse recepti e ter plaatse Paranimfen Paranimfen Faiza El Amrani ([email protected])Faiza El Amrani ([email protected]) Gerard Graat Gerard Graat ([email protected]) Mohsin El Amrani ([email protected]) Mohsin El Amrani Mohsin El Amrani [email protected] [email protected] 2020 2020 Cover.indd 1 4-3-2020 15:53:28 Cover.indd 1 9-3-2020 12:54:39 Quantification of therapeutic antibodies and endogenous proteins with LC-MS/MS Thesis Versie 38 without cover.indd 1 9-3-2020 15:10:38 © 2020 Mohsin El Amrani Cover image: Adobe Stock Lay-out and design by: M. El Amrani Printed by: Ridderprint ISBN: 978-94-6375-730-0 El Amrani, M Quantification of therapeutic antibodies and endogenous proteins with LC- MS/MS All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic, or mechanical, including photocopy, recording, or any information storage or retrieval system, without permission or writing from the author. The copyright of articles that have been published or accepted for publication has been transferred to the respective journals Thesis Versie 38 without cover.indd 2 9-3-2020 15:10:38 Quantification of therapeutic antibodies and endogenous proteins with LC-MS/MS Het kwantificeren van therapeutische antilichamen en endogene eiwitten met LC-MS/MS (met een samenvatting in het Nederlands) PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. H.R.B.M Kummeling, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op donderdag 28 mei 2020 des middags te 2.30 uur door Mohsin El Amrani geboren op 18 september 1978 te Utrecht Thesis Versie 38 without cover.indd 3 9-3-2020 15:10:38 Promotoren Prof. dr. Alwin D.R. Huitema Prof. dr. C. Erik Hack Copromotor Dr. Erik M. van Maarseveen Thesis Versie 38 without cover.indd 4 9-3-2020 15:10:38 Table of Contents Chapter 1 General introduction, scope and outline of the thesis 7 Chapter 2 Therapeutic antibody quantification 2.1 Six-step workflow for the quantification of therapeutic monoclonal 15 antibodies in biological matrices with liquid chromatography mass spectrometry – A Tutorial. 2.2 Quantification of active infliximab in human serum with liquid 43 chromatography-tandem mass spectrometry using a tumor necrosis factor alpha-based pre-analytical sample purification and a stable isotopic labeled infliximab bio-similar as internal standard: A target-based, sensitive and cost-effective method. 2.3 Simultaneous quantification of free adalimumab and infliximab in 61 human plasma using a target based sample purification and liquid chromatography-tandem mass spectrometry. 2.4 Quantification of T-cell binding polyclonal rabbit anti-thymocyte 79 globulin in human plasma with liquid chromatography tandem mass-spectrometry 2.5 Quantification of total dinutuximab concentrations in 99 neuroblastoma patients with liquid chromatography tandem mass spectrometry. Chapter 3 Endogenous protein quantification 3.1 Quantification of neutralizing anti-drug antibodies and their 119 neutralizing capacity using competitive displacement and tandem mass spectrometry: Infliximab as proof of principle. 3.2 Quantification of total coagulation factor VIII in human plasma 135 with liquid chromatography tandem mass spectrometry using immunoaffinity purification. Thesis Versie 38 without cover.indd 5 9-3-2020 15:10:38 Chapter 4 General discussion and future perspectives 153 Summary 165 Samenvatting 173 Dankwoord 181 List of co-authors 185 List of publications 189 About the author 191 Thesis Versie 38 without cover.indd 6 9-3-2020 15:10:38 General introduction, scope and outline of the thesis Thesis Versie 38 without cover.indd 7 9-3-2020 15:10:39 Chapter 1 8 Thesis Versie 38 without cover.indd 8 9-3-2020 15:10:39 General introduction, scope and outline of the thesis Background Quantitative proteomics entails the quantification of endogenous and exogenous therapeutic proteins and plays an important role in disease diagnosis, pharmacokinetics (PK) research, therapeutic drug monitoring and biomarker discovery [1-3]. Therapeutic proteins are widely used in treatment of many diseases such as genetic disorders, inflammatory autoimmune diseases and cancer. Genetic disorders are caused by gene mutations and lead to the expression of a wrongly 1 sequenced protein that is unable to perform its designated task resulting in an overall underexpression of functional protein. Patients can be treatment for low protein expression through intravenous infusion with a recombinant form of the therapeutic protein [4-6]. Quantitative proteomics provides a means to diagnose the disease and its severity, study PK and to monitor the patient during treatment [7]. Moreover, quantitative proteomics is frequently used in biomarker discovery. Indeed, overexpression of proteins can be a good indicator of the presence of certain diseases. For example, elevated levels of prostate-specific antigen can be a sign for prostate cancer, elevated levels of C-reactive protein can be an indicator for heart disease and overexpression of tumor necrosis factor alpha (TNF-α) can be a sign of inflammatory autoimmune disease such as rheumatoid arthritis, Crohn’s disease or ulcerative colitis [8-10]. Protein overexpression in some cases is treated by blocking the disease inducing protein, as in the treatment of patient with TNF-α overexpression with infliximab or adalimumab. During treatment, therapeutic drug monitoring of these therapeutic proteins is strongly recommended to determine drug plasma levels since low levels are associated with the development of anti-drug antibodies and poor treatment outcome [10]. These anti-drug antibodies can also be monitored in plasma and can be used to optimize patient treatment plan [11]. Furthermore, biomarker discovery on cancer cells can also lead to the identification of over-expressed protein which can be ideal targets for monoclonal antibody based therapy [12]. Example of these is the discovery of over-expressed human epidermal growth factor receptor 2 in certain types of breast cancer which can be treated with trastuzumab or over- expressed GD2 receptor on cancer cells in children with neuroblastoma which is treated with dinutuximab [13, 14]. Traditionally, the quantification of endogenous or exogenous therapeutic proteins is performed by ligand binding assays such as Enzyme Linked Immunosorbent Assay (ELISA) or Radioimmunoassay (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 9 often generate different results depending on the specificity and avidity of the antibody Thesis Versie 38 without cover.indd 9 9-3-2020 15:10:39 Chapter 1 used in the test [15]. 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 liquid chromatography-tandem mass spectrometry (LC-MS/ MS) can solve many of the above mentioned limitations found in ligand binding assay and provides additional advantages, such as enhanced sample through-put, linear dynamic range, precision, selectivity and multiplexing abilities [16-19]. Objectives of the thesis The aim of this thesis is to provide insight to the development of LC-MS/MS methods for the quantification of endogenous proteins and exogenous therapeutic proteins such as monoclonal antibodies. These methods lay down the foundation of quantitative proteomics with LC-MS/MS and can serve as templates for future method development. Outline of the thesis Chapter 2: Therapeutic antibody quantification Chapter 2.1 provides a tutorial and an overview of published methods for top down, middle down, and bottom-up quantitative proteomics and explains in six steps how to develop a LC- MS/MS method for the quantification of therapeutic proteins based on the popular bottom- up proteomics approach. Considerations for signature peptide selection are provided and critical method parameters are discussed for internal standard selection, chromatographic separation, sample purification, digestion and method validation. Chapter 2.2 provides an example of the selective purification of active infliximab in human plasma followed by LC-MS/MS quantification. In chapter 2.3, multiplexing is introduced for the simultaneous quantification of both