SCiEX has produced a technical note describing an immunoaffinity-high resolution accurate mass assay for the pre-clinical quantification of Trastuzumab in rat plasma.
LC-MS/MS based assays have seen wide adoption for the quantification of therapeutic peptides and proteins in biological matrices. The typical procedure for a protein therapeutic is enzymatic digestion and quantification of either universal or signature peptides depending on the matrix background. In recent years immunoaffinity sample preparation techniques for sample purification and enrichment before digestion have been widely investigated. There are multiple reasons for the implementation of hyphenated immunocapture-LC-MS/MS. One of the key benefits is the significant increase in selectivity and sensitivity that can be achieved by combining the two techniques in addition to wide dynamic range and excellent robustness and reproducibility.
Immunoaffinity sample prep techniques can be adapted to different assay requirements and different matrices by changing the mode of capture. The mode of capture chosen for the therapeutic protein can be generic when paired with LC-MS/MS, which can be used to get an assay up and running rapidly, or very specific if required. This allows an assay to be adapted through the lifetime of the therapeutic as it transitions from discovery to the clinic.
The generic approach can be utilized in the pre-clinical setting for fast method deployment. For example, using anti-human IgG (Fc specific) capture antibodies allows for a simple and generic methodology for monoclonal antibodies such as Trastuzumab. In addition, multiple candidates can be rapidly screened using the same capture process.
Routinely, immunoaffinity prepared samples are analysed by signature peptide quantification on triple quadrupoles. In this study, we present an immunoaffinity high resolution accurate mass (HRAMS) assay as a complementary analytical technique for signature peptide quantification The assay performance is measured using Trastuzumab in rat plasma as a case study.
This technical note will cover:
- Key challenges in the quantification of Trastuzumab
- Key advantages using immunoaffinity enrichment coupled with MRMHR on the TripleTOF® 6600 System
- Description on how an immunoaffinity-HRAMS assay with the TripleTOF 6600 system can meet the demands of a pre-clinical biotherapeutic assay.