B-204 Analytical Characterization and Stability Assessment of Recombinant Human UCH-L1 for Linearity, Quality Control, and External Quality Assessment Applications

Abstract

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a clinically utilized biomarker of neuronal injury, including traumatic brain injury. Broader implementation of UCH-L1 testing across immunoassay platforms requires well-characterized recombinant materials to support assay linearity verification, internal quality control (QC), and external quality assessment (EQA) programs. Critical attributes include high purity, lot-to-lot consistency, commutability, and statistically justified shelf-life assignment. We describe the expression, purification, analytical characterization, multi-platform performance, and stability assessment of recombinant human UCH-L1 developed for in vitro diagnostic applications. Recombinant human UCH-L1 was expressed in a bacterial expression system and purified using affinity chromatography followed by polishing steps to minimize host cell proteins and aggregates. Purity was assessed by SDS-PAGE, and protein concentrations were measured using the Bradford method and clinical protein assays (Roche). Purified UCH-L1 was formulated into matrix-based preparations for: (1) multi-level linearity panels spanning the analytical measuring range and (2) internal QC materials. Multi-platform analytical performance was evaluated across multiple commercially available immunoassay systems (Roche, Biomerieux, Abbott) using dilutional linearity, recovery, and parallelism assessments. Parallelism assessments confirmed that the recombinant UCH-L1 produced proportional, dose-dependent responses across serial dilutions, supporting consistent performance across the assay’s measuring range. Real-time and accelerated stability studies were conducted under controlled storage conditions. Shelf-life estimation was performed using statistical analysis and methodologies in accordance with CLSI EP25 guidance. Regression modeling was applied to characterize degradation kinetics and project expiration dating. Comparisons between time points were evaluated at a 95% confidence interval using paired and unpaired t-tests, as appropriate. Accelerated data were used to support interpretation of trends but not as the primary basis for shelf-life assignment. The purified recombinant UCH-L1 achieved >95% purity with low aggregate levels. Analytical characterization confirmed molecular identity across production lots. Linearity panels demonstrated strong analytical performance across platforms (R² > 0.99), with acceptable recovery and dilutional linearity throughout the claimed measuring range. QC materials exhibited minimal between-lot variability and stable target value assignment. Real-time stability data demonstrated low-rate, predictable degradation within predefined acceptance limits. Statistical comparisons at the 95% confidence level showed no clinically significant differences within the labeled storage period. Observed real-time stability aligned with projected expiration dating. Recombinant human UCH-L1 can be reproducibly expressed in a bacterial system and purified to high analytical purity using affinity chromatography–based processes. The material demonstrates robust multi-platform compatibility and suitability for linearity verification and QC applications. Based on these characteristics, recombinant UCH-L1 is expected to be compatible for future EQA applications, although formal evaluation is ongoing, providing a pathway to standardized materials for inter-laboratory harmonization of UCH-L1 testing. Stability assessment using CLSI EP25–aligned real-time extrapolation with 95% confidence supports reliable shelf-life assignment and enhances analytical standardization and inter-laboratory harmonization of UCH-L1 testing.