25 Tutorial on LC-MS/MS methods quantifying mAbs INTRODUCTION Therapeutic monoclonal antibodies (mAbs) nowadays are widely accepted as valuable treatment options for patients suffering from a variety of diseases, particularly in the areas of oncology and immune diseases. At present, 76 mAbs have been granted market authorization by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) and are now commercially available for therapeutic use [1]. Judging from drug pipelines this number is set to grow considerably in the near future [2]. Therapeutic mAbs target pathological processes with high specificity and concomitantly lead to fewer side effects compared to conventional small molecule based therapies [3]. Furthermore, due to the high molecular weight of mAbs, the clearance pathway is not by renal elimination after hepatic enzyme metabolism but rather by proteolytic catabolism, receptor-mediated uptake and degradation, and sometimes by the catabolic pathway of their molecular target. Two thirds of mAbs are salvaged from degradation by binding to the protective neonatal Fc-receptor (FcRn) particularly on endothelial cells, which extends their elimination half-life to ~18-21 days [4]. MAb production and design has made great strides from the early discovery in 1975 by Kohler and Milstein [5]. The progression from murine mAbs (1975) using hybridoma technology to chimeric mAbs (1984) using recombinant DNA techniques to humanized mAbs (1988) using complementary determining region (CDR) grafting and finally to fully human mAbs (1994) using phage display or transgenic mice took less than 20 years [6-9]. These steps were essential to reduce the risk of anti-drug antibodies (ADA) development and allergic reactions associated with first generation mAbs [10-12]. In fact, additional requirements from the EMA, Food and Drug Administration (FDA) and World Health Organization (WHO) for the evaluation and monitoring of immunogenicity of new biopharmaceuticals were mandated as part of regulatory approval, together with a rigorous post-authorization pharmacovigilance with product-level traceability for of all biopharmaceuticals [13-17]. The discovery of new therapeutic targets and the high treatment efficacy of biopharmaceuticals accelerated the development of novel mAbbased therapies [16, 18]. For this purpose, bioanalytical methods were necessary to facilitate the required preclinical pharmacokinetic (PK) studies. In addition, therapeutic drug monitoring of mAbs concentrations can highlight accelerated drug clearance in patients which is indicative of ADA development and loss of drug response. Traditional bioanalytical methods such as ligand-binding assays rely on an anti-idiotypic antibody or a ligand with high avidity towards the therapeutic protein of interest. However, the development of such antibodies is notoriously difficult and time consuming [19-21]. Therefore, advances in analytical techniques were essential to attain shorter method development times, which is why liquid chromatography tandem mass-spectrometry (LC-MS/MS) has received increasing interest as an alternative method for quantification over the last decade. Following strength and weaknesses analysis of ligand binding assays, this tutorial systematically addresses bioanalytical methods to quantify therapeutic mAbs in biological matrices using LC-MS. Three main branches of quantitative proteomics 2
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