REVIEW ARTICLE Practical Considerations for the Pharmacokinetic and Immunogenic Assessment of Antibody–Drug Conjugates Melody Sauerborn • William van Dongen Ó Springer International Publishing Switzerland 2014 Abstract Currently, the most bioanalytically challenging drugs are antibody–drug conjugates (ADCs), constructs comprising a monoclonal antibody and a cytotoxic drug connected by a linker. The bioanalytical challenges arise from the heterogeneous nature of ADCs and their complex in vivo behavior, resulting in a high number of analytes to be measured. Measuring the concentration of biologics in blood/plasma/serum is a necessity to properly assess their pharmacokinetic (PK)/pharmacodynamic behaviors in vivo. An additional bioanalytical challenge is to monitor the stability of the ADCs, as cytotoxic drugs released from the ADC in blood circulation may pose a potential safety risk because of their high cytotoxic potency. The nature of ADCs does not only complicate bioanalysis, but also immunogenicity assessment. Questions, such as ‘Which part of the ADCs is the anti-drug antibodies directed against?’ may arise, and their answer normally includes several immunogenicity risk assessment strategies. This review will focus on the bioanalytical challenges of ADCs, current approaches involving ligand-binding assays (LBAs), liquid chromatography and mass spectrometry platforms, and recommendations on which approach to use for which stage of drug development, and will close with immunogenicity assessment. In order to appropriately tackle the bioanalytical and immunogenic challenges of ADCs and consider every angle, the authors of this review have expertise in ligand binding and liquid chromatogra- phy–mass spectrometry. Key Points Antibody–drug conjugates (ADCs) are heterogeneous in nature, and therefore their pharmacokinetic (PK) and immunogenic assessments are challenging. Bioanalytical platforms are in most cases a combination of LBAs and liquid chromatography– mass spectrometry in order to evaluate all aspects of an ADC, including the small and large molecule parts. With increasing numbers of ADCs in clinical trials and post-marketing, data will be generated that will guide both industry and regulatory agencies to more defined guidelines and/or white papers about the assessment of ADC PK and immunogenicity. 1 Introduction In the fight against cancer, conventional treatments either include cytotoxic drugs or monoclonal antibodies (mAbs) [1]. Cytotoxic drugs are very efficient in killing tumor cells but can have unwanted off-target effects due to a lack of specific targeting moieties. mAbs are very target specific, and most of their anti-tumor effects are mediated by their fragment crys- tallizable (Fc) part by two main mechanisms: antibody- dependent cellular cytotoxicity and complement-dependent cytotoxicity. Nevertheless, mAbs alone lack the high potency of cytotoxic drugs. In order to achieve high efficacy and specificity, the two mechanisms of action were combined via synthetic linkers to form antibody–drug conjugates (ADCs) M. Sauerborn (&) Á W. van Dongen Department of Bioanalysis, TNO Triskelion, Utrechtsweg 48, 3704 HE Zeist, The Netherlands e-mail: melody.sauerborn@tno.triskelion.nl BioDrugs DOI 10.1007/s40259-014-0096-z