Screening Non-neutralizing Anti-idiotype Antibodies Against a Drug Candidate for Total Pharmacokinetic and Target Engagement Assay.

Non-neutralizing anti-idiotype antibodies against a therapeutic monoclonal antibody (mAb) play a crucial role in the creation of total pharmacokinetic (PK) assays and total target engagement (TE) assays during both pre-clinical and clinical development. The development of these anti-idiotype antibodies is challenging. In this study, we utilized a hybridoma platform to produce a variety of anti-idiotype antibodies against GSK2857914, a humanized IgG1 anti-BCMA monoclonal antibody. The candidate clones were evaluated using surface plasmon resonance (SPR) and bio-layer interferometry (BLI) for binding affinity, binding profiling, matrix interference, and antibody pairing determination. We discovered that three anti-idiotype antibodies did not prevent BCMA from binding to GSK2857914. All three candidates demonstrated high binding affinities. One of the three exhibited minimal matrix inference and could pair with the other two candidates. Additionally, one of the three clones was biotinylated as a capture reagent for the total PK assay, and another was labeled with ruthenium as a detection reagent for both the total PK assay and total TE assay. The assay results clearly show that these reagents are genuine non-neutralizing anti-idiotypic antibodies and are suitable for total PK and TE assay development. Based on this and similar studies, we conclude that the hybridoma platform has a high success rate for generating non-neutralizing anti-idiotype antibodies. Our methodology for developing and characterizing non-neutralizing anti-idiotype antibodies to therapeutic antibodies can be generally applied to any antibody-based drug candidate's total PK and total TE assay development.

Intact mAb LC-MS for drug concentration from pre-clinical studies: bioanalytical method performance and in-life samples.

Background: Antibody biotherapeutic measurement from pharmacokinetic studies has not been traditionally based on intact molecular mass as is the case for small molecules. However, recent advancements in protein capture and mass spectrometer technology have enabled intact mass detection and quantitation for dosed biotherapeutics. A bioanalytical method validation is part of the regulatory requirement for sample analysis to determine drug concentration from in-life study samples. Results/methodology: Here, an intact protein LC-MS assay is subjected to mock bioanalytical method validation, and unknown samples are compared between intact protein LC-MS and established bioanalytical assay formats: Ligand-binding assay and peptide LC-MS/MS. Discussion/conclusion: Results are presented from the intact and traditional bioanalytical method evaluations, where the in-life sample concentrations were comparable across method types with associated data analyses presented. Furthermore, for intact protein LC-MS, modification monitoring and evaluation of data processing parameters is demonstrated.