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Published on: March 24, 2015
Validation parameters for a novel biosensor assay which simultaneously measures serum concentrations of a humanized
1Schering-Plough Research Institute, MS K-15 / 2700, Kenilworth, NJ 07033, USA.
Insights
This study validates a BIAcore assay for quantifying humanized monoclonal antibodies in mouse serum. The assay also detects and characterizes anti-drug antibodies, requiring minimal sample volume and offering automation.
Area of Science:
- Biotechnology
- Immunology
- Assay Development
Background:
- Biosensor assays are crucial for drug development and characterization.
- Quantifying monoclonal antibodies and detecting anti-drug antibodies in preclinical studies is essential.
- Existing methods may require large sample volumes or lack automation.
Purpose of the Study:
- To validate a novel BIAcore assay for simultaneous quantitation of humanized monoclonal antibodies and detection of anti-drug antibodies in mouse serum.
- To assess key assay validation parameters including precision, accuracy, linearity, stability, specificity, and sensitivity.
- To demonstrate the assay's utility with small sample volumes and its automation capabilities.
Main Methods:
- A tandem assay format using a BIAcore 2000 instrument was developed.
- Flowcells were sequentially coated with the target antigen and the humanized monoclonal antibody (mAb).
- Mouse serum samples were analyzed for mAb quantitation and anti-mAb antibody detection.
Main Results:
- The assay accurately quantifies humanized mAb in mouse serum with a limit of quantitation of 1 microg/ml.
- The assay successfully detects and characterizes anti-humanized mAb antibodies by isotype.
- The assay requires only 10 microliters of serum and is amenable to automation.
Conclusions:
- The validated BIAcore assay provides a precise, accurate, and sensitive method for analyzing humanized monoclonal antibodies and anti-drug antibodies in mouse serum.
- The assay's small sample requirement and automation capability offer significant advantages for preclinical research.
- This method facilitates individual sample analysis, reducing the need for pooled samples.
Abstract:
This report documents the validation of an assay using BIAcore 2000 that, with a single injection of mouse serum, allows the quantitation of a humanized monoclonal antibody and can also detect the presence of antibodies directed against this humanized antibody. The important components required for the validation of biosensor assays including precision, accuracy, linearity, stability of the immobilized ligand, specificity and sensitivity are addressed. The tandem assay that is used as a model for biosensor validations is accomplished by flowing each sample across the surface of two flowcells in sequence. The first flowcell has the antigen that the humanized mAb was generated against immobilized while the humanized mAb itself is immobilized on the second flowcell. The quantitation component of this assay is precise and accurate with a limit of quantitation of 1 microg/ml in mouse serum samples. Any antibodies directed against the humanized mAb can be detected and also characterized as to isotype. This unique assay can be performed with as little as 10 microl of serum which is then diluted ten-fold prior to analysis. The small sample requirement allows analysis of individual mouse serum samples rather than requiring the use of pooled samples from multiple mice. A further advantage of this assay is the automation capability which allows unattended operation.
