Premium
Benchmarking a fluorescence microplate‐based aggregation assay using subvisible protein particle standards
Author(s) -
Shen Dee,
Chan Eric,
Dai Lijun,
Coleman Jack,
Patton Wayne F
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.909.11
Subject(s) - chemistry , fluorescence , chromatography , particle aggregation , protein aggregation , bradford protein assay , particle (ecology) , nanoparticle , nanotechnology , materials science , biochemistry , physics , oceanography , quantum mechanics , geology
Aggregation represents one of the most significant obstacles to protein‐based pharmaceutical development. During drug formulation, aggregation can impact product quality in terms of biological activity and immunogenicity. Improved methods are required to detect and monitor aggregation during manufacturing. We report upon a fluorescence microplate assay employing a novel fluorescent probe specifically devised to detect aggregated proteins and peptides. The probe is nonfluorescent in solution, but becomes brightly fluorescent when bound to the tertiary structure of aggregated proteins. The assay is capable of detecting less than 0.2% aggregated protein in a concentrated antibody formulation, with a linear dynamic range of two‐orders of magnitude. Sub‐visible oligomeric particles are readily detected without a need for sample separation, solvent exchange or dilution. Subvisible protein particle standards were developed to benchmark the fluorescence microplate assay, as well as orthogonal characterization methods, including laser‐based nanoparticle tracking and micro‐flow Imaging. Advantages of the microplate‐based assay include simple implementation with widely available instrumentation, results achievable in 30 minutes or less and robust assay performance. The assay provides a convenient, complementary orthogonal method for cross‐validation of instrument‐intensive techniques. This work was supported by ENZO Life Sciences.