Scientific Associate Director Takeda Pharmaceuticals Burlington, Massachusetts
Enzymatic activity assays conducted in support of long-term cell and gene therapy clinical trials can vary in cost, complexity and performance due to reagent performance and handling as well as reagent and sample stability. These challenges are difficult to resolve without the availability of an enzymatic reference standard. In addition, assay complexity as well as enzyme stability in matrix make traditional strategies for proving assay reproducibility problematic.
To ensure continuity in enzymatic activity data over time, bulk reagents and bulk 4-Methylumbelliferone calibration standards were prepared and qualified in the presence of freshly prepared calibration standards and quality controls (QCs) of known performance. The bulk materials were then stored at nominal -80°C for long-term use to reduce variability during periodic enzymatic activity measurements. Feasibility testing of frozen 4-MU calibrators was initiated to overcome variation in daily preparations attributed to analyst technique differences, diluent pH differences and 4-MU handling/stability as well as lot-to-lot variation. Due to lack of proven long-term storage stability of critical and non-critical reagents, assay performance was monitored by trending assay performance indicators over time such as instrument responses and back-calculated activity of pre-screened, frozen endogenous QCs as well as freshly prepared recombinant enzyme QCs. Frozen curve instrument responses were also measured over time to evaluate changes in calibrators relative to performance observed upon initial qualification. To confirm the trending strategy of 4-MU in frozen diluent, low and high calibrator points were stored long-term at nominal -80°C, then tested against fresh 4MU calibrators. These results will be discussed to provide generalized approaches and considerations for long-term data continuity between patients and their respective time points. Presentation Description: Conditionally activated bispecific T cell engagers (TCEs) activates T‐cells through the creation of artificial immune synapses in the tumor microenvironment (TME). Their pharmacological response involves the dynamic inter‐relationships among T‐cells and surface receptors on the tumor cells. However, the presence of a domain with high immunogenicity risk may result in an overall high immunogenicity risk level for the entire multi-domain biotherapeutic and may negatively impacts pharmacokinetics (PK), pharmacodynamics (PD), safety, and efficacy of these drugs. Ligand-binding assays (LBAs) in bridging format are typically used for PK and for anti-drug antibody (ADA) measurements of large molecule therapeutics. We initially developed a free PK assay using reagents specific to the functional domains. Anti-drug antibodies (ADAs) specific to these functional domains can interfere in the ligand binding assay by competing for epitopes recognized by the assay antibodies or by preventing assay antibody binding through steric hindrance. This can lead to underestimation of total drug concentration in pharmacokinetic (PK) samples which can confound the understanding of the exposure response relationship and critical decisions during drug development. When reagent options are limited due to specificity or binding regions this can present significant challenges in mitigating ADA tolerance of the PK assay.
We have developed a novel approach using immunoaffinity coupled with LC-MS\MS (or hybrid LBA LC\MS) to meet the challenges associated with multi-domain drug PK measurements in biological matrices that contain high levels of ADA’s. This presentation will use real-world examples and case studies that will highlight actual biological and analytical challenges and subsequently showcase the utility of hybrid LBA LC\MS to help understand and overcome these challenges.
This talk will also show how a series of bioanalytical assays including free and total PK assays, domain specific ADA and Nab assays, and PD markers are used in combination to elucidate the exposure response relationship for a highly complex therapeutic modality.
Learning Objectives:
After this talk audience will know about a novel bioanalytical assay including free and total PK assay overcoming the ADA interference to elucidate the exposure response relationship.
This presentation will use real-world examples and case studies that will highlight analytical challenges and subsequently showcase the utility of hybrid LBA LC\MS to help understand and overcome these challenges.
Audience will know about a novel assay that we developed using immunoaffinity coupled with LC-MS\MS to meet the challengeswith multi-domain drug PK measurements that contain high levels of ADA’s