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

• Published in: Journal of pharmaceutical and biomedical analysis 2013-05, Vol.78-79, p.224-232
• Main Authors: Moberg, Anna, Lager, Anna, Hämäläinen, Markku D., Jarhede, Tanja
• Format: Article
• Language: English
• Subjects: Anti-drug antibodies (ADAs); Biosensing Techniques; biosensors; Blood Chemical Analysis; blood serum; experimental design; immune response; Immunogenicity; Limit of Detection; Multivariate Analysis; Multivariate design of experiments (DoE); Non-specific binding from plasma; screening; sodium chloride; surface plasmon resonance; Surface plasmon resonance (SPR) assay; Surface Plasmon Resonance - methods; Therapeutic antibody
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2013.02.018

[Display omitted] ► Efficient parallel experiments using DoE gave increased sensitivity in immunogenicity assays. ► Immobilization level and salt concentration influenced sensitivity most. ► Buffer with 500mM NaCl reduced background binding and variability from plasma. ► Optimal assay conditions gave detection levels in the range of 80–170ngml−1. The sensitivity of biosensor assays in complex media such as plasma or serum is often limited by non-specific binding. The degree of binding often varies between individuals and therefore a large number of different plasma samples have to be used during assay development. Some surface plasmon resonance (SPR) biosensors allow for parallel screening of several running buffer compositions, with a number of different immobilization levels for each buffer. These technical possibilities combined with statistical design of experiments (DoE) enable efficient parallel optimization of multiple assay conditions. In this paper we outline how to increase the sensitivity in SPR-based assays by minimizing background binding and variability from negative control plasma while retaining high signals from positive samples. To mimic immunogenicity studies of biotherapeutics we have used a model assay with anti-rituximab as an anti-drug antibody to be detected in plasma by binding to immobilized rituximab. Immobilization level and sodium chloride concentration were found to be the most important factors to optimize. There were also a number of significant interaction effects and strong non-linearites between the buffer composition/immobilization level and the assay performance, which necessitated DoE based optimization strategies. The applicability of the optimized conditions was verified with several assays (anti-erythropoietin, omalizumab, anti-IgE and anti-myoglobin) in spiked plasma samples resulting in detection levels in the range of 80–170ngml−1. The buffer conditions presented in this paper can be used for other immunogenicity assays on biosensor platforms or as a good starting point for further assay development for new immunogenicity assays.