Increased sensitivity of SPR assays in plasma through efficient parallel assay optimization

Area of Science:

• Biotechnology
• Analytical Chemistry
• Immunology

Background:

• Biosensor assay sensitivity in complex media (plasma, serum) is often hindered by non-specific binding.
• Variability in binding across individuals necessitates extensive plasma sample use during assay development.
• Surface plasmon resonance (SPR) biosensors offer parallel screening of buffer compositions and immobilization levels.

Purpose of the Study:

• To enhance SPR-based assay sensitivity by minimizing background binding and variability in negative control plasma.
• To maintain high signals from positive samples in biotherapeutic immunogenicity studies.
• To optimize assay conditions using statistical design of experiments (DoE) for efficient parallel optimization.

Main Methods:

• Utilized a model assay detecting anti-rituximab in plasma using immobilized rituximab.
• Employed design of experiments (DoE) to optimize multiple assay conditions in parallel.
• Investigated immobilization level and sodium chloride concentration as key optimization factors.

Main Results:

• Immobilization level and sodium chloride concentration were identified as critical optimization parameters.
• Significant interaction effects and non-linearities between buffer composition, immobilization level, and assay performance were observed.
• Optimized conditions were validated across multiple assays (anti-erythropoietin, omalizumab, anti-IgE, anti-myoglobin) in spiked plasma, achieving detection levels of 80-170 ng/mL.

Conclusions:

• The presented buffer conditions and optimization strategy effectively minimize background binding and variability in SPR immunogenicity assays.
• The optimized conditions provide a robust starting point for developing new immunogenicity assays on biosensor platforms.
• This approach significantly improves the sensitivity and reliability of detecting anti-drug antibodies in complex biological matrices.