Validation of a microfluidic platform to measure total therapeutic antibodies and incurred sample reanalysis performance.

BACKGROUND: A microfluidic platform-based assay was validated to measure a humanized or fully human IgG in rat serum samples. MATERIALS & METHODS: The cumulative assessment for accuracy and precision was performed with three accuracy and precision runs. RESULTS: The inter-assay accuracy (mean %bias) ranged from -4.3 to 3.8%, and inter-batch %CV ranged from 5.0 to 9.2%. The method acceptance criterion was determined as 15% total error. The assay dynamic range was 50 to 10000 ng/ml. Incurred sample reanalysis passed with 95% of samples meeting incurred sample reanalysis acceptance criteria. Potential carryover effect was not observed. CONCLUSION: This study demonstrated the need for evaluating additional platform-specific processes when new technologies are employed to ensure the reproducibility of a successfully validated microfluidic platform method.

Impact of anti-drug antibodies in preclinical pharmacokinetic assessment.

The administration of human biotherapeutics is often associated with a higher incidence of immunogenicity in preclinical species. The presence of anti-drug antibodies (ADAs) in the test samples can affect the accurate measurement of therapeutic protein (TP) in bioanalytical methods designed to support pharmacokinetic (PK) and toxicokinetic (TK) assessments. The impact can vary depending on the bioanalytical method platform and study dosing design. The goal of this study is to evaluate the impact of ADA response on the bioanalytical methods in support of PK/TK and the associated study data interpretation. Sprague Dawley rats were administered with four weekly doses of 50 mg/kg TP, a humanized monoclonal antibody. The TP in serum samples was measured using three bioanalytical methods that quantified bound and/or unbound TP to ADA. The ADA response in the animals was classified into negative, low, medium, and high based on the magnitude of the response. The presence of ADA in samples led to discrepant TP measurements between the methods, especially at time points where the TP concentrations were low. This could be due to ADA interference to the accurate measurement of ADA-bound TP concentrations. The TP concentration at last time point (C last) was reduced by 82.8%, 98.6%, and 99.8%, respectively, for samples containing low, medium, and high levels of ADA. The interfering effects of the ADA on bioanalytical methods and exposure were evident as early as 2 weeks post-dosing. This modeling approach can provide the better understanding of ADA impact on PK exposure in multiple doses.

Model-based strategy for bioanalytical method comparison: measurement of a soluble ligand as a biomarker.

Ligand binding assays (LBAs) are often the method of choice for quantification of protein biomarkers and therapeutic biologics during drug development. Soluble ligand X is a glycoprotein. To understand the role of circulating ligand X in drug-target relationship, an analytical method (Method 1) was developed and validated to measure circulating ligand X and to support early clinical studies. Change in the detection reagent led to the development and validation of a second method (Method 2). Both methods measure total circulating ligand X levels. To ensure that the method specificities and data were consistent upon method change, the two methods were cross-validated using three distinct sample types: (1) recombinant ligand X (rLIGX) spiked in buffer, (2) authentic serum samples containing endogenous ligand X (eLIGX), and (3) serum samples collected from patients being dosed with the therapeutic antibody (incurred samples). Methods were considered comparable if the 90% confidence interval (90% CI) fell within 0.80-1.25 for all sample types. The results from the comparison reveal that two methods were comparable for rLIGX samples with the 90% CI of 0.90-1.07. However, with eLIGX samples, Method 1 produced higher mean (± SD) concentrations 675 (± 316 pg/mL) than Method 2 195 (± 97 pg/mL) and the two methods were considered not comparable as the 90% CI was 0.27-0.29. With the incurred samples, the comparison results also indicated the incomparability of these two methods as the 90% CI was 0.57-0.65. To describe the statistically relevant relationship between two methods in analyzing the serum samples, linear and quadratic regression models were applied to derive two conversion equations; one each for eLIGX and incurred samples. The applicability of the equations was verified with independent study data to indicate that the equations can be used to relate two different sets of study data. A model-based strategy presented here can serve as an explicatory paradigm for other analogous situations in the future.