Enrichment and Liquid Chromatography–Mass Spectrometry Analysis of Trastuzumab and Pertuzumab Using Affimer Reagents

Abstract

Trastuzumab and pertuzumab are monoclonal antibodies used in the treatment of human epidermal growth factor receptor-2 (HER2)-positive breast cancer. Therapeutic proteins may undergo chemical modifications that may affect the results of bioanalytical assays, as well as their therapeutic efficacy. Modifications may arise during production and storage, as well as after administration to patients. Studying in vivo biotransformation of monoclonal, therapeutic antibodies requires their enrichment from plasma to discriminate them from endogenous antibodies, as well as from other plasma proteins. To this end, we screened Affimer reagents for selectivity toward trastuzumab or pertuzumab. Affimer reagents are alternative binding proteins possessing two variable binding loops that are based on the human protease inhibitor stefin A or phytocystatin protein scaffolds. Affimer reagents were selected from an extensive library by phage display. The four best-performing binders for each therapeutic antibody were prioritized using a microtiter plate-based approach combined with liquid chromatography–mass spectrometry (LC–MS) in the selected reaction monitoring (SRM) mode. These Affimer reagents were immobilized via engineered 6-His or Cys tags to Ni 2+ - or maleimide beads, respectively. Recovery values of 70% and higher were obtained for both trastuzumab and pertuzumab when spiked at 100, 150, and 200 μg/mL concentrations in human plasma followed by trypsin digestion in the presence of 0.5% sodium deoxycholate and 10 mM dithiothreitol (DTT). Notably, the maleimide beads showed undetectable unspecific binding to endogenous immunoglobulin G (IgGs) or other plasma proteins when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enrichment method was applied to samples from stress tests of the antibodies at 37 °C to mimic in vivo conditions.

Probing Molecular Interactions of Antibody Drugs, Silicone Oil, and Surfactant at Buried Interfaces In Situ

“…Antibodies play essential roles in the immune system of a human body, which can bind to specific antigens with high efficiencies and selectivities. In the recent years, antibody molecules including lab-produced antibodies have been widely used as drugs to treat a variety of diseases including cancer. , Especially recently, therapeutic antibodies have been developed to treat COVID-19 patients. , The function of an antibody is heavily dependent on its structure, including its dominant secondary structures. It is therefore critical to study antibody structures in different conditions to understand the structure–function relations, providing knowledge on how drug efficiency can be improved based on structure optimization.…”

“…In the recent years, antibody molecules including lab-produced antibodies have been widely used as drugs to treat a variety of diseases including cancer. 1,2 Especially recently, therapeutic antibodies have been developed to treat COVID-19 patients. 3,4 The function of an antibody is heavily dependent on its structure, including its dominant secondary structures.…”

Pertuzumab Charge Variant Analysis and Complementarity-Determining Region Stability Assessment to Deamidation

“…51 In this study, we have applied an anti-pertuzumab Affimer to enrich pertuzumab and its different forms from patient plasma samples. 22 The enriched pertuzumab was analyzed by peptide mapping after enzymatic digestion. The results of the analysis of patient samples indicated that in vivo biotransformation can be mimicked by stressing in vitro.…”

Large Molecule Bioanalysis by LC–MS: Beyond Simply Quantifying

“…This means, for example, that if a given target protein used for screening does not contain the recognition element that is needed for following an in vivo biotransformation, it will be impossible to find an appropriate binder for the biotransformation product. An Affimer reagent that was selected because of its good binding properties toward trastuzumab showed a significant decrease in extraction recovery when trastuzumab was incubated at pH 7.4 and 37 • C for 3 weeks to mimic in vivo conditions, while a similar treatment of pertuzumab did not lead to reduced extraction efficiency by its own optimal Affimer [17]. This could be due to a much-reduced recognition by the trastuzumab binder of the various biotransformation products that were formed upon incubation, while the pertuzumab binder might still be able to capture the modified forms of this biopharmaceutical if these were formed at all.…”