@article {Olaleye249, author = {Oladapo Olaleye and Baubek Spanov and Peter Bults and Anna van der Voort and Natalia Govorukhina and Gabe S. Sonke and Peter Horvatovich and Nico C. van de Merbel and Rainer Bischoff}, title = {Biotransformation of Trastuzumab and Pertuzumab in Breast Cancer Patients Assessed by Affinity Enrichment and Ion-Exchange Chromatography}, volume = {51}, number = {2}, pages = {249--256}, year = {2023}, doi = {10.1124/dmd.122.001094}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {Therapeutic proteins (TPs) are known to be heterogeneous due to modifications that occur during the production process and storage. Modifications may also occur in TPs after their administration to patients due to in vivo biotransformation. Ligand binding assays, which are widely used in the bioanalysis of TPs in body fluids, are typically unable to distinguish such modifications. Liquid chromatography coupled to mass spectrometry is being increasingly used to study modifications in TPs, but its use to study in vivo biotransformation has been limited until now. We present a novel approach that combines affinity enrichment using Affimer reagents with ion-exchange chromatography (IEX) to analyze charge variants of the TPs trastuzumab and pertuzumab in plasma of patients undergoing therapy for HER2-positive breast cancer. Affimer reagents were immobilized via engineered Cys tags to maleimide beads, and the TPs were eluted under acidic conditions followed by rapid neutralization. The enriched TPs were analyzed by cation-exchange chromatography (IEX) using pH-gradient elution, resulting in the separation of about 20 charge variants for trastuzumab and about five charge variants for pertuzumab. A comparison between in vitro stressed TPs spiked into plasma, and TPs enriched from patient plasma showed that the observed profiles were highly similar. This indicates that in vitro stress testing in plasma can mimic the situation in patient plasma, as far as the generation of charge variants is concerned.SIGNIFICANCE STATEMENT This research attempts to elucidate the modifications that occur in therapeutic proteins (TPs) after they have been administered to patients. This is important because there is little knowledge about the fate of TPs in this regard, and certain modifications could affect their efficiency. Our results show that the modifications discovered are most likely due to a chemical process and are not patient specific.}, issn = {0090-9556}, URL = {https://dmd.aspetjournals.org/content/51/2/249}, eprint = {https://dmd.aspetjournals.org/content/51/2/249.full.pdf}, journal = {Drug Metabolism and Disposition} }