Large Molecule Bioanalysis: Quantitative Analysis of Monoclonal Antibodies by LC-MS/MS

The study of protein function in genomics and proteomics has accelerated our understanding of protein function in disease processes. This new knowledge has expanded the possibilities for intervention by protein drugs. Monoclonal antibodies (MAb), chimeric or fusion proteins, and gene therapy are being explored for therapeutic application. Preclinical and clinical studies require assay methods for such protein drugs, be they manufactured or expressed. Unfortunately, classical ligand binding assays such as ELISA do not always meet researchers’ needs.

QPS has developed a general work flow for the assay of monoclonal antibody drugs by LC-MS/MS (Figure 1). In the first step, the total antibody fraction of a plasma or serum sample is isolated by size exclusion clean-up. In the second step, all protein in the fraction is enzymatically digested to afford a mixture of peptides. The last step comprises LC-MS/MS analysis for the detection of one or more specific peptides from the particular antibody drug. This work flow supports rapid method development, as it avoids the use of any target specific reagents.

Typically, method development starts with a computer-assisted evaluation of the protein drug amino acid sequence. This assessment reveals potential digest peptides that may distinguish the protein drug from other proteins in the matrix—for example, by comparison to all known sequences from the human genome. A first LC-MS/MS analysis of a neat therapeutic MAb digest is then used to characterize these potential target peptides. Chromatographic and mass spectrometric behaviors are used in the selection of one or more peptides to afford the desired selectivity and sensitivity. After this establishment of target peptides, method development and validation can be conducted pretty much as with small molecule LC-MS/MS assays.

Figure 2 shows a sample result for the assay of trastuzumab in human plasma at 2.5 µM—typical for the low end of a required MAb assay range. This demonstrates that the approach provides a suitable determination method for MAb.

Fig 1. QPS' generic work flow for the bioanalysis of monoclonal antibody drugs by LC-MS/MS

Fig 2. Trastuzumab LC-MS/MS bioanalysis using the molecule's unique peptide DTYIHWRV, demonstrating the generic approach's feasibility

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QPS Netherlands 
Ben van Baar, PhD; Senior Scientist
Jaap Wieling, PhD; VP of Bioanalysis and Technology R&D.

20 years in pharma R&D navigation