Abstract
In this study, we developed a generic bioanalytical workflow providing sensitive, specific, and accurate absolute quantification of therapeutic monoclonal antibodies in serum. The workflow involves magnetic beads coated with protein A to pull-down therapeutic monoclonal antibodies with affinity for protein A from the biological matrix, followed by tryptic digestion and LC-MS/ MS quantification of a unique signature peptide, considering of course the matrix of interest and other present mAbs, if applicable. The feasibility of this approach was demonstrated for Inflix imab (trade name Remicade) in rat serum. The assigned signature peptide was monitored in the selected reaction monitoring (SRM) mode. Assay variability was determined to be below 20%, except at the QC low level, which was provided through optimization of the sample preparation and monitoring of the LC-MS/MS using a stable isotope labeled signature peptide as internal standard. The 100 ng/ml lower limit of quantification using only 25 µl sample volume, is generally considered as sufficient for pharmaceutical development purposes for monoclonal antibodies.
Highlights
In order to quantify proteins in complex biological matrices such as serum or plasma, LC-MS/MS is finding its way to the bioanalytical community [1,2,3,4,5,6] as a complementary method to ligand binding assays (LBA)
We developed a generic workflow consisting of immunoprecipitation, using protein A capture followed by LC–MS/MS quantification of a unique signature peptide after tryptic digestion of the target therapeutic monoclonal antibody in biological matrices [11]
There are several other relevant peptide selection criteria, which were not found to be relevant for Infliximab and were not applied
Summary
In order to quantify proteins in complex biological matrices such as serum or plasma, LC-MS/MS is finding its way to the bioanalytical community [1,2,3,4,5,6] as a complementary method to ligand binding assays (LBA). In contrast to LBAs, the conventional standard for therapeutic protein bioanalysis, which measures the unbound intact 3D do-. J. APPL.BIOANAL munoglobulins it is suitable to concentrate therapeutic monoclonal antibodies which are usually immunoglobulin based. To demonstrate the feasibility of this approach, Infliximab (trade name Remicade), a chimeric monoclonal antibody against tumor necrosis factor alpha (TNF-α) used to treat autoimmune diseases, was used [13]
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