Improvement of drug tolerance in immunogenicity testing by acid treatment on Biacore.

Detection of anti-drug antibodies (ADA) can be difficult, if not impossible, in the presence of drug in the sample. This is a particular concern with therapeutic monoclonal antibodies (mAbs), which have typically longer half-lives than other proteins. For detection of ADA in presence of high drug concentrations, assay choice is limited to ELISA-like methods, capable of incorporating acid dissociation procedures to separate drug-ADA immune complexes. To our knowledge, Biacore assays have not been shown to be directly compatible with acid dissociation procedures, until now. As a consequence, steps to ensure adequate clearance of the drug are prerequisite to enable sensitive detection of ADA. Here we describe the development of a novel, rapid and highly drug tolerant Biacore method that uses an acid dissociation step to detect ADA in the presence of excess drug in human serum. Removal of drug after acid treatment is not required.

Protein microarray platform for the multiplex analysis of biomarkers in human sera.

Many biomarkers are currently used to monitor patients in clinical studies. Technologies which evaluate, validate and monitor biomarkers in a cost effective and efficient manner are a necessity. Here we describe the development, validation and implementation of a protein microarray platform for the quantitative and simultaneous analysis of six proteins: IL-1beta, IL-1ra, IL-6, IL-8, MCP-1 and TNFalpha. The platform utilizes a 96-well plate as a solid support on which antibodies are immobilized using non-contact piezoelectric printing. The reaction is based on a sandwich ELISA and the signal is quantified by chemiluminescence with a CCD camera. The robustness and reproducibility of the methodology was investigated using the Food and Drug Administration (FDA) regulatory guidelines for pharmacokinetic assay validation, in which a spike-recovery validation test was elaborated and run over 3 days. The method was shown to be both quantitative and reproducible, with assay accuracy between 70% and 130%, and an assay precision of less than 30%. In addition, protein microarray performance was compared with the classical ELISA approach. Sera collected from a total of 78 individuals were assayed using both approaches. Correlation coefficients (R2) between the two technologies were calculated for each of the analytes: 0.90 for IL-1beta, 0.60 for IL-1ra, 0.93 for IL-6, 0.96 for IL-8, 0.94 for MCP-1 and 0.95 for TNFalpha. The results obtained demonstrate the applicability of this protein microarray for quantitative and simultaneous analysis of IL-1beta, IL-1ra, IL-6, IL-8, MCP-1 and TNFalpha in clinical samples.