Establishing Measurement Assurance via Interlaboratory Characterization: Consensus Physical Titer for Candidate Reference Materials

Published

February 23, 2026

Author(s)

Jamie Almeida, Edward Kwee, Lili Wang, Sheng Lin-Gibson

Abstract

Introduction Gene therapy manufacturing requires standardized analytical characterization of viral and non-viral vectors to assess gene delivery system quality. Lack of reproducibility in physical titer measurements makes product comparability and regulatory filing challenging. To address these challenges, the NIST Flow Cytometry Standards Consortium (FCSC) Working Group 4 (Gene Delivery Systems) initiated an interlaboratory study (ILS) to assess reproducibility and comparability across conventional and novel methods for physical titer measurements. Methods Candidate commercial reference materials contributed by consortium members include three adeno-associated virus serotypes, three lentivirus materials, adenovirus, lipid nanoparticle, and extracellular vesicle materials. An expert committee comprising government, industry, and academic stakeholders developed harmonized protocols for genome titer, capsid titer, and particle concentration assays. Genome titer protocols were developed by the ILS committee to address and reduce sources of variability by incorporating control samples. The committee also incorporated best practices from literature and member organizations that had not been assessed in a large-scale interlaboratory study format. Standardized ELISA protocols were implemented using manufacturer provided protocols to evaluate capsid titer. Capsid titer controls were included to assess variability of the ELISA kits. Particle concentration measurement methods include nanoparticle flow cytometry, resistive pulse sensing, mass photometry, and nanoparticle tracking analysis to assess metrics correlation with traditional titer methods. Characterization is ongoing for the interlaboratory study materials. Results Standardized protocols for AAV and LVV genome titer were successfully developed and tested. Using these protocols, the study assessed variation between measurement replicates and homogeneity across multiple sample vials. For AAV contributions, vial-to-vial variation ranged between 0.1% and 5%, while LVV contributions exhibited a wider variation of 2% to 25%. Particle concentration assessments included rigorous evaluation of measurement replicates, day-to-day variation, and linearity between dilutions to define the effective linear range for each method. When comparing these orthogonal measurement methods, large differences were observed within the tested samples. These differences suggest varying purity levels in the materials, highlighting the different measurands that orthogonal physical titer methods are targeting. Conclusions The NIST FCSC WG4 interlaboratory study is assigning consensus titer values to candidate reference materials and provide a level of measurement assurance not possible through a single organization. The resulting validated protocols will be made available to the community to enable global reproducibility and comparability. This work provides a critical understanding of how emerging particle concentration technologies correlate with conventional titer measurements, facilitating the adoption of new in-process analytics in gene therapy manufacturing.

Proceedings Title

NIIMBL National Meeting 2026

Conference Dates

June 23-25, 2026

Conference Location

Washington DC, DC, US

Conference Title

NIIMBL National Meeting

Pub Type

Conferences

Citation

Almeida, J. , Kwee, E. , Wang, L. and Lin-Gibson, S. (2026), Establishing Measurement Assurance via Interlaboratory Characterization: Consensus Physical Titer for Candidate Reference Materials, NIIMBL National Meeting 2026, Washington DC, DC, US (Accessed February 24, 2026)