At a glance
A global biopharma sponsor developing a novel bispecific nanobody needed a fit-for-purpose PK assay that could accurately measure drug concentrations with high sensitivity.
In order to achieve this, Syngene redesigned the dynamic range of the curve to 1 ng/ml and successfully delivered clinical PK data for dose selection. The assay repeat rate was 1% after overcoming challenges with assay drift and a systemic negative bias in the quality control performance.
Background
A global biopharma sponsor was developing a novel bispecific nanobody. These compact antibody-derived proteins are engineered to bind two different targets simultaneously. Their small size, high specificity, and faster systemic clearance compared to conventional monoclonal antibodies make them attractive for targeted therapies, but they also create unique demands on bioanalytical methods.
For this program, the sponsor needed a pharmacokinetic (PK) assay that could quantify nanobody concentrations across very low and very high exposure levels in clinical samples. The assay had to be adequately sensitive to measure terminal-phase concentrations while robustly handling peak levels after dosing, with minimal re-analysis and consistent performance across study runs.
Challenge
During early method development, the ligand-binding assay demonstrated few challenges:
- The transferred method did not provide the required dynamic range for low concentration measurements.
- There was assay drift on the plate, which impacted the sensitivity of the method.
- When regressed, the assay demonstrated a systemic negative bias.
- These issues increased the risk of repeat analysis with an impact on assay sensitivity, which would render the PK data unreliable for clinical decision making.
Syngene’s solution
Syngene’s large molecule bioanalytical team implemented a stepwise experimental approach to stabilize the method and extend its dynamic range to achieve a fit-for-purpose assay.
First, the standard curve was redesigned. By optimizing calibration levels and the signal response, the dynamic range was extended from 1.00 to 1000 ng/mL. This allowed accurate quantification of high and low nanobody concentrations in the same run, thus reducing the need for dilutions or repeat testing.
Next, the team addressed assay drift. Signal trends, instrument response, and spike-recovery data were reviewed to identify contributions from reagent use and plate handling. Two levers were then adjusted:
- Titration of key reagents, including capture and detection components, to provide a more stable signal across the plate.
- Refined mixing and loading patterns, with standardized mixing and revised well-loading sequences to minimize temporal drift and edge effects.
With these changes, spike-recovery bias improved from a wide range of –15% to –65% to a narrow band around –4% to –8%, appropriate for a fit-for-purpose PK assay.
Clinical application and PK profiling
After optimization, the method was validated and applied to clinical samples. The wider dynamic range of 1.00 to 1000 ng/mL enabled accurate measurement of high exposure samples without compromising sensitivity. Drug levels as low as 1.43 ng/mL were reliably detected, demonstrating adequate sensitivity of the method to measure end-of-trial concentrations.
Across the study, the repeat rate was ~1%, reflecting robustness and a lesser need for re-analysis. The resulting concentration–time profiles across subjects were smooth and internally consistent, supporting estimation of exposure parameters such as Cmax and AUC for clinical and regulatory discussions.
Outcome and sponsor value
The optimized assay gave several benefits to the sponsor and to the study. It extended the usable range from 1.00 to 1000 ng/mL, improved accuracy, with spike-recovery results mostly between –4% and –8% of the nominal value. The method could reliably detect drug levels as low as 1.43 ng/mL and showed a low repeat rate of ~1%, which reduced data reporting time and sample use. By fixing the assay challenges and handling development, validation, and clinical sample analysis within the same team, Syngene generated robust PK data that supported dose selection and further clinical planning for the bispecific nanobody.
Syngene’s Bioanalytical Capabilities
Syngene provides advanced bioanalytical laboratory solutions for biologics, offering large molecule bioanalysis for monoclonal antibodies, bispecific antibodies, nanobodies, fusion proteins, and other complex biologics. Our team develops and validates fit-for-purpose methods for PK/PD, immunogenicity, and biomarker assays, delivering regulated bioanalysis under GLP compliance. These services support translational research, preclinical toxicology, first-in-human studies, and Phase I–III clinical trials. Sponsors can rely on Syngene as a single partner for bioanalytical testing and clinical trial support, helping to speed up development and strengthen data for decision-making.