LC−MS/MS Approach for Quantification of Therapeutic Proteins in Plasma Using a Protein Internal Standard and 2D-Solid-Phase Extraction Cleanup

Abstract

The bioanalysis of plasma samples generated from in vivo studies of therapeutic proteins is of increasing interesting in the biopharmaceutical industry. The conventional ELISA approach has a long assay development time which can limit use in the early discovery and development of protein-based drugs. In this study, an LC-MS/MS bioassay was developed for the quantification of somatropin and a therapeutic human monoclonal antibody. The assay used bovine fetuin as an internal standard and a two-dimensional solid-phase extraction for the cleanup of the plasma digest. Sample extracts were resolved on an analytical size column using a 6 min LC gradient and analyzed using a triple-quadruple mass spectrometer. The linearity of the assay for somatropin was established from 1 to 1000 microg/mL with accuracy and precision within 15%. This LC-MS approach was also applied to a rat pharmacokinetic study of the therapeutic monoclonal antibody with a lower quantitation limit of 0.5 microg/mL. The LC-MS assay had improved accuracy and precision, and the results from analysis of in vivo study samples showed good agreement with the data obtained with an ELISA. The results from this study indicate that the LC-MS bioassay is a simple and feasible approach for the bioanalysis of therapeutic proteins to support in vivo studies during early drug discovery and development.

Recommendations for Validation of LC-MS/MS Bioanalytical Methods for Protein Biotherapeutics

“…Successful examples have been reported using molecules with appropriate properties (14,17). For example, selection of similar size surrogate peptides of multiple mAb analytes at the same framework location led to similar digestion and chromatographic behaviors for bioanalysis of the mAbs from a cassette dosing study (14).…”

“…As a general rule, it is recommended to use the simplest approach that will achieve the required selectivity/specificity, sensitivity, accuracy, and precision in the intended matrix and species. Approaches can range from traditional sample preparation (e.g., protein precipitation or solid phase extraction) to affinity capture enrichment strategies and from generic to specific LC-MS/MS assays (12)(13)(14)(15)(16)(17). Depending upon assay requirements, a protein therapeutic may be analyzed following simple direct This white paper may also provide useful principles for validating LC-MS/MS-based assays for other types of biotherapeutics, including undigested peptides and proteins, antibody-drug conjugates (ADCs), and other hybrid proteinbased biotherapeutics.…”

The Bottleneck in the Cancer Biomarker Pipeline and Protein Quantification through Mass Spectrometry–Based Approaches: Current Strategies for Candidate Verification

“…proteins in both single (33)(34)(35)(36)(37)(38)(39)(40) and multiplexed assay formats (41)(42)(43)(44). The proteins quantified in these studies are summarized in Table 1.…”

Quantification of growth hormone in serum by isotope dilution mass spectrometry

“…However, in spite of earlier model experiments to extend the scope of the technique to proteins [7] it was not until recently that quantification by IDMS of protein markers in body fluids for diagnostic purposes has been reported [8][9][10][11][12][13]. Measurement of GH by MS has been discussed previously, namely in a feasibility study [14], a discussion of MS as fast alternative method regarding the time needed for development of antibody based assays [15] and with an investigation about optimal conditions for proteolysis of serum samples [16]. Neither of the methods was applicable to measurement of samples in clinical practice at primary level, however, since no use was made of isotopically labeled internal standards and concentration ranges considered in these examples were far above what is relevant in clinical practice.…”